Novel antagonist antibodies and their fab fragments against gpvi and uses thereof

ABSTRACT

The present invention discloses novel antibodies that specifically bind to the human platelet membrane protein Glycoprotein VI (GPVI) and their monovalent fragments or derivatives. The antibodies of the invention are antibodies from hybridoma clone 390 and fragment antibodies thereof able to induce a GPVI depletion pheno-type. These antibodies and Fab fragments are able to block collagen binding and thus preventing platelet activation by collagen.  The invention also relates to hybridoma clones and expression plasmids for the production of disclosed antibodies and Fab fragments. The present invention further refers to the uses of monovalent antibody fragments to manufacture research, diagnostic and immunotherapeutic agents for the treatment of thrombosis and other vascular diseases. The invention also concerns a Fab bearing  a molecule at the C-terminal extremity, as well as method for prevention of recognition of Fab by antibodies using such modified Fab. The invention concerns a method for prevention of platelet activation when an anti-GP VI Fab is used.

FIELD OF THE INVENTION

The present invention discloses novel antibodies that specifically bindto the human platelet membrane protein Glycoprotein VI (GPVI) and theirmonovalent fragments or derivatives. The antibodies of the invention areantibodies from hybridoma clone 390 and antibodies that bind to anepitope which is similar to the conformational epitope recognized by theantibody from hybridoma clone 390. These antibodies and Fab fragmentsare able to block collagen binding and thus preventing plateletactivation by collagen. The invention also relates to hybridoma clonesand expression plasmids for the production of disclosed antibodies andFab fragments. The present invention further refers to the uses ofmonovalent antibody fragments to manufacture research, diagnostic andimmunotherapeutic agents for the treatment of thrombosis and othervascular diseases. In another aspect, the invention concerns a method toprevent receptor activation through patient specific anti-Fab antibodiesinduced receptor clustering.

BACKGROUND OF THE INVENTION

Platelet function is of fundamental importance in the development ofarterial thrombosis and cardiovascular diseases. Nowadays it is a matterof course that patients suffering from cardiovascular diseases aretreated with antiplatelet drugs. Despite the availability of variousclinically successful antiplatelet therapies, there is still a largeunmet medical need for new treatments. This deficiency is mainly causedby the limited efficacy of the currently available drugs, particularlyin regard to the drugs efficacy—safety (bleeding) correlation.Interfering with early events of platelet activation and adhesion, amechanism not targeted by drugs currently in use, would be an attractiveapproach for the improvement of the efficacy—safety margin. The collagenreceptor glycoprotein (GPVI) is of central importance in these earlyevents of platelet activation, and therefore a major target for theinterference with this mechanism (Nieswandt B and Watson S P, Blood.2003 Jul. 15;102(2):449-61). The antiplatelet and antithrombotic effectsof GPVI have been described in several in vitro and in vivo systems,using platelets from mice and men. Platelets deficient in GPVI arerendered unresponsive to collagen, one of the most importantthrombogenic components of the subendothelial matrix (Lockyer S. et al,Thromb Res. 2006; 118(3):371-80). Moreover, mouse studies have shownthat GPVI deficiency causes an effective inhibition of arterial thrombusformation at the damaged vessel wall without increasing thesusceptibility to spontaneous bleeding. All these data suggest that GPVIrepresents an effective and safe target for the treatment of arterialthrombosis. The central role of GPVI in the initiation of thrombusformation indicates that inhibition of this receptor may be beneficialin syndromes of arterial thrombosis. This makes the use ofbiotherapeutic proteins such as antibodies a clinically meaningfulstrategy for the inhibition of GPVI. All the more since the interactionof GPVI and its ligand collagen seems to involve an expanded proteinsurface, a successful interference with this protein-protein interactionis more likely with inhibitory GPVI-binding proteins as compared toother strategies. An in vivo proof of concept for the inhibition of GPVIfunction by antibodies and Fab fragments has been shown in severalanimal models.

There is still a need for clinically effective inhibitors of GPVIactivity.

GPVI is a major collagen receptor expressed exclusively on platelets andmegacaryocytes. Binding to collagen induces receptor clustering andsubsequent platelet activation. This is one of the initial events inthrombus formation. Current anti-platelet drugs interfere with thrombusformation through targeting late events in this process. A serious sideeffect of these drugs is prolonged bleeding which limits their use.There are several lines of evidence that targeting early events inthrombus formation (such as GPVI) can be highly antithrombotic without amajor impact on bleeding liability. The interaction between collagen andGPVI can be successfully inhibited by neutralizing monoclonal antibodies(mAb). However as mAb's are bivalent molecules, they can induceGPVI-receptor clustering and therefore platelet activation. Tocircumvent this, monovalent antibody fragments such as Fab fragmentshave been developed.

Unfortunately, in depth safety profiling of these monovalent anti-GPVIFab fragments reveals a potential to still induce platelet activation ina patient specific manner. To offer a safe therapeutic for the treatmentof ischemic events, this activatory potential of the developed Fabmolecules had to be abolished. Until now, this problem has not beenresolved.

SUMMARY OF THE INVENTION

In a first aspect, this invention provides a novel monoclonal antibody(from clone 390) which specifically binds to human GPVI, as well as toprimate GPVI.

In another aspect, the invention concerns recombinant Fab fragmentsderived from the variable domain sequences of anti-GPVI mAb from clone390. These anti-GPVI antibody and Fab fragments recognize a specificconformational epitope in D1 and D2 domains of the human GPVI protein.

Both the anti-GPVI mAb and the Fab fragments are able to inhibitcollagen binding to GPVI. The anti-GPVI Fab fragments have beenhumanized and engineered with the aim to improve their affinity to humanGPVI. The biophysical characteristics of the humanized variants aredescribed.

In addition to the inhibition of collagen, the anti-GPVI Fab fragmentsare able to inhibit platelet aggregation induced by collagen both inhuman platelet rich plasma and in human whole blood. These Fab Fragmentsare also able to inhibit the thrombus formation under flow on a collagencoated surface.

Thus, inhibition of GPVI by antibodies in humans appears as anattractive antithrombotic strategy.

Surprisingly, the anti-GPVI Fab of the invention induces a GPVIdepletion phenotype. Thus, anti-GP VI Fab fragments able to induce GPVIdepletion phenotype constitute another object of the invention.

The invention also provides a method for prevention of recognition ofFab fragments by pre-existing antibodies consisting in masking theC-terminal extremity of the Fab by addition of a molecule. The inventionalso provides a method for prevention of recognition of Fab fragments bynew antibodies directed toward Fab C-terminal extremity consisting inmasking the C-terminal extremity of the Fab by addition of a molecule.

The invention also provides a method for prevention of plateletactivation when an anti-GPVI Fab is used, where the C-terminal extremityof the Fab is masked by addition of a molecule.

Another object of the invention is a Fab fragment bearing a molecule atthe C-terminal extremity.

DNA and protein sequences as well as vectors for expression of theanti-GPVI and Fab fragments of the invention are also provided.

The antithrombotic agents of the invention may be used for the treatmentof thrombotic or vascular diseases.

The invention also provides an antithrombotic composition comprising apharmaceutically effective amount of a GPVI specific monoclonal antibodyfragment of the invention with appropriate excipients.

In another aspect of the invention, the antibodies can be used fordiagnosis of patients at risk requiring anti-thrombotic treatment. Theinvention also encompasses a kit for diagnosis including anti-GPVIantibodies or fragments thereof.

The invention also provides a method for the preparation of GPVIantibody, antibody fragments and masked antibody Fab fragments of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, this invention provides a novel monoclonal antibodyand fragments thereof which specifically binds to human GPVI, inparticular the anti-GPVI mAb from hybridoma clone 390. The antibodies ofthe present invention antagonize, totally or partially, the activity ofGPVI.

In a second aspect of the invention, the inventors have demonstratedthat the addition of a peptide at the C-terminal extremity of the heavychain (HC) of a Fab has a masking effect that avoids recognition of Fabmolecules by preexisting anti-Fab antibodies in some patients andtherefore prevents platelet activation and associated side effects.

As used herein, “monoclonal antibody from hybridoma clone 390” or“monoclonal antibody from clone 390” refers to an antibody defined bySEQ ID NO.6 and SEQ ID NO.8 as well as its derivatives includinghumanized antibodies, Fab fragments and humanized Fab fragments or anyimproved version of these molecules especially as described in thepresent application.

The term “antibody” is used herein in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD andIgE, polyclonal antibodies, multispecific antibodies, chimericantibodies, and antigen binding fragments. An antibody reactive with aspecific antigen can be generated by recombinant methods such asselection of libraries of recombinant antibodies in phage or similarvectors, or by immunizing an animal with the antigen or anantigen-encoding nucleic acid.

A typical IgG antibody is comprised of two identical heavy chains andtwo identical light chains that are joined by disulfide bonds. Eachheavy and light chain contains a constant region and a variable region.Each variable region contains three segments called“complementarity-determining regions” (“CDRs”) or “hypervariableregions”, which are primarily responsible for binding an epitope of anantigen. They are usually referred to as CDR1, CDR2, and CDR3, numberedsequentially from the N-terminus. The more highly conserved portions ofthe variable regions are called the “framework regions”.

In a particular embodiement, the antibodies and fragments thereof of theinvention, including the antibody Fab fragment, comprise the 6 CDRdefined by SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQID NO:22 and SEQ ID NO:23.

In another embodiement, the antibodies and fragments thereof of theinvention, including the antibody Fab fragment, comprise the 6 CDRdefined by SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:38, SEQ ID NO:21, SEQID NO:39 and SEQ ID NO:23.1n another particular aspect, the antibody Fabfragment of the invention comprises:

-   -   (a) complementarity determining regions (CDRs) of a heavy chain        variable region (HCVR) having amino acid sequences defined by        SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 (or SEQ ID        NO:38); and    -   (b) complementarity determining regions (CDRs) of a light chain        variable region (LCVR) having amino acid sequences defined by        SEQ ID NO: 21, SEQ ID NO: 22 (or SEQ ID NO:39), and SEQ ID NO:        23; and    -   wherein at least 2 amino acid residues of each CDR can be        changed to another amino acid residue without directly        disrupting a contact with a GPVI epitope residue.

As used herein, “directly disrupting a contact with the GPVI epitoperesidue” refers to changing an antibody amino acid residue that is incontact with a GPVI epitope residue and described in the crystalstructure of the examples provided herein.

As used herein, “VH” refers to the variable region of an immunoglobulinheavy chain of an antibody, including the heavy chain of an Fv, scFv,dsFv, Fab, Fab′ or F(ab′)2 fragment. Reference to “VL” refers to thevariable region of the immunoglobulin light chain of an antibody,including the light chain of an Fv, scFv, dsFv, Fab, Fab' or F(ab′)2fragment.

A “polyclonal antibody” is an antibody which was produced among or inthe presence of one or more other, non-identical antibodies. In general,polyclonal antibodies are produced from a B-lymphocyte in the presenceof several other B-lymphocytes producing non-identical antibodies.Usually, polyclonal antibodies are obtained directly from an immunizedanimal.

A “monoclonal antibody”, as used herein, is an antibody obtained from apopulation of substantially homogeneous antibodies, i.e. the antibodiesforming this population are essentially identical except for possiblenaturally occurring mutations which might be present in minor amounts.These antibodies are directed against a single epitope and are thereforehighly specific.

As used herein, the terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, include anynaturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. Antigen-binding fragments of an antibody maybe derived, e.g., from full antibody molecules using any suitablestandard techniques such as proteolytic digestion or recombinant geneticengineering techniques involving the manipulation and expression of DNAencoding antibody variable and optionally constant domains. Non-limitingexamples of antigen-binding fragments include: (i) Fab fragments; (ii)F(ab′)₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v)single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimalrecognition units consisting of the amino acid residues that mimic thehypervariable region of an antibody (e.g., an isolated complementaritydetermining region (CDR)). Other engineered molecules, such asdiabodies, triabodies, tetrabodies and minibodies, are also encompassedwithin the expression “antigen-binding fragment,” as used herein.

As used herein, the term “Fab fragment” corresponds to the light chain(LC) plus part of the heavy chain (HC) of the antibody. Fab moleculesare produced either by proteolytic cleavage of IgG molecules or madethrough expression of recombinant molecules. Usually parts of theconstant domain of the heavy chain (Fc part) are removed.

As used herein, the term “masked Fab fragment” or “masked Fab”corresponds to a Fab fragment containing the light chain (LC) plus partof the heavy chain (HC) of the antibody and where the C-terminus of theHC has been extended by a peptide to mask recognition of the Fab bypreexisting antibodies in a patient.

The antibodies of the invention have been obtained by the generation ofhybridoma through mouse immunization technique using a human recombinantGPVI protein containing only extracellular D1 and D2 domains. Thesequences of the protein used for immunization correspond to SEQ ID NO.3and SEQ ID NO.4.

The obtained mAbs have been selected first for their ability torecognize the human GPVI protein, but also for their capability to blockthe binding of collagen to the human GPVI protein and to bind the GPVIprotein with a high affinity.

The antibodies show cross-reactivity to primate GPVI (as shown in FIG.2) whereas no cross-reactivity exists against murine, rat, dog, swine orrabbit GPVI.

The selected mAb is able to bind to the human GPVI protein with a pMaffinity (as shown in Table 1) and to block collagen binding to thehuman GPVI protein with IC50 values of less than 10 μg/mL (as shown inFIG. 1A). This antibody is also able to block collagen binding tonon-human primate GPVI (as shown in FIG. 1B).

In another aspect, the invention concerns recombinant Fab fragmentsderived from the variable domain sequences of anti-GPVI mAb from clone390. Fab fragments can be prepared by any method know in the art. Forexample proteolytic Fab fragments can be obtained by Ficin cleavage orrecombinant Fab fragments can be prepared by cloning the requiredsequences into an expression vector (for example as described in Example3B). Like the anti-GPVI mAb, the Fab fragments are able to inhibitcollagen binding to GPVI.

The Fab fragments have been co-crystallized with GPVI and analyzed byX-ray crystallography to determine the epitope recognized by the Fab onthe GPVI protein. This analysis allowed defining a conformationalepitope involving both D1 and D2 domains in the extracellular domain ofthe human GPVI. The numbering system used in the eptiope corresponds toGPVI with the 20 residue signal sequence removed and as shown in FIG. 16and SEQ ID NO:32.

In one aspect of the invention, an isolated antibody or antigen bindingfragment that specifically binds a conformational epitope of human GPVIand contacts human GPVI residues comprising Ser 43, Arg 67 and Asp 81.In another aspect of the invention, the antibody or antigen bindingfragment makes additional contacts with two or more GPVI residuesselected from the group consisting of Pro4, Lys7, Glu40, Lys41, Leu42,Ser44, Ser45, Arg46, Arg65, Trp76, Leu78, Pro79, Ser80, Gln82, Ser165,and Arg166. In another aspect of the invention, the antibody or antigenbinding fragment makes additional contacts with three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, ten or more, eleven or more, twelve or more, thirteen or more,fourteen or more, fifteen or more GPVI residues selected from the groupconsisting of Pro4, Lys7, Glu40, Lys41, Leu42, Ser44, Ser45, Arg46,Arg65, Trp76, Leu78, Pro79, Ser80, Gln82, Ser165, and Arg166.

As used herein, “contacting residues” are defined as residues with adistance of less than 4.5 A to GPVI antigen residues or the reverse andas measured in a crystal structure between an antibody to GPVI orantigen binding fragment of the antibody and a GPVI antigen using asoftware program such as NCONT of the CCP4 Software package or anequivalent software program.

In one embodiement, the antibody Fab fragment binds a conformationalepitope of human GPVI and contacts human GPVI residues comprising Ser43, Arg 67 and Asp 81.

In another embodiement, the antibody Fab fragment binds a conformationalepitope of human GPVI and contacts human GPVI residues comprising:

Pro4, Lys7, Glu40, Lys41, Leu42, Ser43, Ser44, Ser45, Arg46, Arg65,Arg67, Trp76, Leu78, Pro79, Ser80, Asp81, Gln82, Ser165, Arg166

The mAb of the invention recognize the same conformational epitope sincethe Fab fragments contain the variable domain sequences of anti-GPVI mAbfrom clone 390.

Thus, in another aspect, the invention provides an anti-GPVI antibodyincluding Fab fragment, which recognizes a conformational epitopecomprised in the extracellular D1 and D2 domains of the human GPVIprotein.

Thus in another aspect, the invention provides a monoclonal anti-GPVIantibody which recognizes a conformational epitope comprising thefollowing amino acids:

Glu40, Lys41, Leu42, Ser43, Ser44, Ser45, Arg46.

In another aspect, the invention provides an anti-GPVI antibody whichrecognizes a conformational epitope comprising the following aminoacids:

Trp76, Leu78, Pro79, Ser80, Asp81, Gln82.

In another embodiment, the anti-GPVI antibody recognizes aconformational epitope comprising the following amino acids:

Glu40, Lys41, Leu42, Ser43, Ser44, Ser45, Arg46, Trp76, Leu78, Pro79,Ser80, Asp81, Gln82.

In another embodiment, the anti-GPVI antibody recognizes aconformational epitope, comprising the following amino acids:

Pro4, Lys7, Glu40, Lys41, Leu42, Ser43, Ser44, Ser45, Arg46, Arg65,Arg67, Trp76, Leu78, Pro79, Ser80, Asp81, Gln82, Ser165, Arg166.

In another embodiment, the anti-GPVI antibody recognizes aconformational epitope consisting in the following amino acids: Pro4,Lys7, Glu40, Lys41, Leu42, Ser43, Ser44, Ser45, Arg46, Arg65, Arg67,Trp76, Leu78, Pro79, Ser80, Asp81, Gln82, Ser165, Arg166.

In a particular embodiement, the antibody Fab fragment of the inventioninvolves mainly D1 domain of the human GPVI.

The antibody of the present invention can be defined as a monoclonalanti-GPVI antibody which (i) competitively inhibits the binding of anantibody comprising a HC of SEQ ID NO.6 and a LC of SEQ ID NO.8 to humanGPVI and (ii) binds to an epitope similar to the conformational epitoperecognized by antibody comprising a HC of SEQ ID NO.6 and a LC of SEQ IDNO.8, or a part of this conformational epitope, with an affinity of atleast 10 nM (KD≦10⁻⁸ M) as determined by a biophysical methods as forexample Surface Plasmon Resonance (Biacore as described in Karlsson R,Larsson A. (2004), J. Mol. Biol. 248, 389-415).

As used herein, the term “K_(D)” refers to the dissociation constant ofa particular antibody/antigen interaction.

The K_(D) reflecting the interaction between the human GPVI protein andthe antibody and Fab of the invention is comprised in the [10⁻⁷; 10⁻¹⁰]interval. An antibody specific for human GPVI present a K_(D)=10⁻⁷ M,preferably a K_(D)=10⁻⁸ M, more preferably a K_(D)=10⁻⁹ M. The antibodywith the highest affinity may have a K_(D)=10⁻¹⁰ M or below, for example10⁻¹¹ M or 10⁻¹²M. The term “specifically binds,” or the like, meansthat an antibody or antigen-binding fragment thereof forms a complexwith a GPVI antigen that is relatively stable under physiologicconditions. Specific binding can be characterized by a dissociationconstant of 1×10⁻⁶ M or less. Methods for determining whether twomolecules specifically bind are well known in the art and include, forexample, equilibrium dialysis, surface plasmon resonance as in Examples2 and 3, and the like. For example, an antibody that “specificallybinds” human GPVI, as used in the context of the present invention,includes antibodies that bind human GPVI or portion thereof with a K_(D)of less than about 1000 nM, less than about 500 nM, less than about 300nM, less than about 200 nM, less than about 100 nM, less than about 90nM, less than about 80 nM, less than about 70 nM, less than about 60 nM,less than about 50 nM, less than about 40 nM, less than about 30 nM,less than about 20 nM, less than about 10 nM, less than about 5 nM, lessthan about 4 nM, less than about 3 nM, less than about 2 nM, less thanabout 1 nM or less than about 0.5 nM, such as measured in a surfaceplasmon resonance assay.

An “epitope” is the site on the antigen to which an antibody binds. Ifthe antigen is a polymer, such as a protein or polysaccharide, theepitope can be formed by contiguous residues or by non-contiguousresidues brought into close proximity by the folding of an antigenicpolymer. In proteins, epitopes formed by contiguous amino acids aretypically retained on exposure to denaturing solvents and are known as“linear epitopes”, whereas epitopes formed by non-contiguous amino acidsare typically lost under said exposure and are known as “conformationalepitopes”.

As used herein, the term “similar epitope” concern a set of amino acidslocated in the same region as the amino acids described bycrystallography analysis as being involved physically in the interactionbetween antibodies derived from clone 390 and the extracellular domainof the human GPVI protein. A similar epitope can include severaldifferences, for example up to five amino acids differences in thevicinity of the epitope of reference. A similar epitope can also presentone or more modifications in the amino acids identified as forming theepitope, i.e. the amino acids interacting with the GPVI protein. One ormore amino acid identified as part of the epitope of reference may beabsent, and one or more additional amino acid residues may be present toform the similar epitope. For example, up to five amino acids may bechanged without affecting the characteristics of the antibody comparedto antibody from hybridoma clone 390. Thus, the region wherein theepitope is located for one specific antibody encompasses somepotentially additional interacting amino acids. In this view, a similarepitope can be defined as a region for competitive binding with theantibody of reference”. When a neutralizing antibody binds to suchregion, one or more cellular signalling pathway(s) of the receptoris/are inhibited leading to a specific impairment of one or morebiological function(s) of the targeted receptor or more generallyeffective protein.

A “neutralizing” or “blocking” antibody, as used herein, is intended torefer to an antibody whose binding to GPVI: (i) interferes with theinteraction between GPVI and collagen, (ii) and/or (iv) results ininhibition of at least one biological function of GPVI. The inhibitioncaused by a GPVI neutralizing or blocking antibody needs not to becomplete as long as it is detectable using an appropriate assay.Exemplary assays for detecting GPVI inhibition are described herein

The existence of topographic regions in proteins linked to specificbiological activities is for example illustrated in patent U.S. Pat. No.6,448,380.

Antibodies recognizing a similar epitope as antibodies derived fromclone 390 can be selected by a competitive ELISA assay using GPVI-Fcfusion protein as capturing antigen. Plates coated with GPVI-Fc fusionprotein are incubated with hybridoma supernatants or antibody or antigenbinding fragments derived from clone 390. A lack of binding of differentanti-GPVI antibodies (labelled by any standard technique) to epitopeblocked GPVI indicates the recognition of a similar epitope. Anequivalent selection strategy can be pursued by competitive Biacoreanalysis in which GPVI is immobilized and the antibody (fragments)derived from clone 390 is used to block the epitope. Other GPVI bindingantibodies candidate are now analysed for binding to epitope blockedGPVI. Thus antibodies that recognize a similar epitope as epitoperecognized by the antibody of the invention can be selected and furthercharacterized by analysis of co-crystal structures using X-ray analysis.

The invention also concerns a humanized and engineered anti-GPVIantibody and fragment thereof.

In the context of the invention, the anti-GPVI Fab fragments have beenhumanized using a method previously described in WO2009/032661, but anyhumanization method known in the art can be used.

Based on the analysis of the crystallographic complex of the Fab withthe GPVI, several mutations have been introduced both for humanizationand with the aim to improve the affinity of the Fab to the human GPVI.

As a result, 3 variants for the LC and 5 variants for the HC have beengenerated. These Fab variants are summarized in Table 6 and in Table 7,respectively for LC (VL) and HC (VH).

Among all possible combinations, the invention concerns in particularthe following combinations:

VL1 with VH1 which correspond to SEQ ID NO.15 and SEQ ID NO.10respectively

VL1 with VH2 which correspond to SEQ ID NO.15 and SEQ ID NO.11respectively

VL1 with VH3 which correspond to SEQ ID NO.15 and SEQ ID NO.12respectively

VL1 with VH4 which correspond to SEQ ID NO.15 and SEQ ID NO.13respectively

VL1 with VH5 which correspond to SEQ ID NO.15 and SEQ ID NO.14respectively

VL2 with VH2 which correspond to SEQ ID NO.16 and SEQ ID NO.11respectively

VL3 with VH2 which correspond to SEQ ID NO.17 and SEQ ID NO.11respectively

VL3 with VH4 which correspond to SEQ ID NO.17 and SEQ ID NO.13respectively

In a preferred embodiment, the humanized variant of anti GPVI Fab isassociation of VL1 with VH3 corresponding to SEQ ID NO.15 with SEQ IDNO.12.

In one aspect of the invention, the VH of the anti-GPVI antibodies canbe further modified by the addition of tags or amino acid extensions(peptide) to mask the antibody from recognition by preexistingantibodies. A sequence of six or more histidine residues, in particular(His)₆, or (His)₇, or (His)₈, or a unit such as GlyGlyGlyGlySer or(GlyGlyGlyGlySer)₂ can be added at the C-terminus of the heavy chain.

As used herein, “engineered Fab Fragment” refers to Fab fragments thathave been genetically engineered to contain a peptide extension of theheavy chain at the c-terminus. The extension causes the previousc-terminus to be obscured and prevents recognition and binding of theFab fragments to pre-existing anti-Fab antibodies.

In another embodiment of the invention, an engineered Fab fragmentcomprising a combination of a humanized heavy chain (HC) amino acidsequence and a humanized light chain (LC) amino acid sequence and ac-terminal extension is provided. The engineered Fab fragment furthercomprises a combination of a heavy chain variable region (HCVR) aminoacid sequence and a light chain variable region (LCVR) amino acidsequence, selected from the group consisting of

-   -   (a) LCVR (SEQ ID NO.15) and HCVR (SEQ ID NO.10);    -   (b) LCVR (SEQ ID NO.15) and HCVR (SEQ ID NO.11);    -   (c) LCVR (SEQ ID NO.15) and HCVR (SEQ ID NO.12);    -   (d) LCVR (SEQ ID NO.15) and HCVR (SEQ ID NO.13);    -   (f) LCVR (SEQ ID NO.16) and HCVR (SEQ ID NO.11);    -   (g) LCVR (SEQ ID NO.17) and HCVR (SEQ ID NO.11); and    -   (h) LCVR (SEQ ID NO.17) and HCVR (SEQ ID NO.13).        wherein the c-terminal extension is selected from the group        consisting of SEQ ID NO:35; SEQ ID NO:36; and SEQ ID NO:37.

In a particular embodiment, the invention concerns a monoclonal antibodycomprising the HC of SEQ ID NO.6 and the LC of SEQ ID NO.8 or a sequencehaving at least 80%, 85%, 90%, 95% or 99% identity with these sequencesbut which retains the same activity as the said monoclonal antibody.

In a more particular embodiement, the antibody Fab fragment of theinvention comprises (a) heavy chain variable region having amino acidsequences defined by SEQ ID NO:6; and (b) light chain variable regionhaving amino acid sequences defined by SEQ ID NO: 8.

The invention also concerns nucleic acids encoding anti-GPVI antibodiesand Fab of the invention. In one embodiment, the nucleic acid moleculeencodes a HC and/or a LC of an anti-GPVI antibody. In a preferredembodiment, a single nucleic acid encodes a HC of an anti-GPVI antibodyand another nucleic acid molecule encodes the LC of an anti-GPVIantibody.

In a particular embodiment, nucleic acids or polynucleotides encodingpolypeptides of the HC and LC from the antibody from hybridoma 390correspond to SEQ ID NO.5 and SEQ ID NO.7 respectively, or a sequencehaving at least 80%, 85%, 90%, 95% or 99% identity with these sequencesbut which retains the same activity as the said monoclonal antibody.

The polynucleotide encoding a polypeptide selected from the groupconsisting in SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ IDNO.16, and SEQ ID NO.17 or a sequence having at least at least 80%, 85%,90%, 95% or 99% identity with these sequences but which retains the sameactivity as the said monoclonal antibody are also part of the presentinvention.

The invention provides vectors comprising the polynucleotides of theinvention. In one embodiment, the vector contains a polynucleotideencoding a HC of an anti-GPVI antibody. In another embodiment, saidpolynucleotide encodes the LC of an anti-GPVI antibody. The inventionalso provides vectors comprising polynucleotide molecules encoding,fusion proteins, modified antibodies, antibody fragments, and probesthereof.

A vector of the invention contains polynucleotides of SEQ ID NO.5 or SEQID NO.7 or any polynucleotide encoding a polypeptide selected from thegroup consisting in SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10,SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15,SEQ ID NO.16, and SEQ ID NO.17 or a sequence having at least at least80%, 85%, 90%, 95% or 99% identity with these sequences but whichretains the same activity as the said monoclonal antibody.

In order to express the HC, fragment of HC and/or LC of the anti-GPVIantibodies or Fab of the invention, the polynucleotides encoding said HCand/or LC are inserted into expression vectors such that the genes areoperatively linked to transcriptional and translational sequences.Expression vectors include plasmids, YACs, cosmids, retrovirus,EBV-derived episomes, and all the other vectors that the skilled manwill know to be convenient for ensuring the expression of said heavyand/or light chains. The skilled man will realize that thepolynucleotides encoding the HC/or HC fragment and the LC can be clonedinto different vectors or in the same vector. In a preferred embodiment,said polynucleotides are cloned in the same vector.

Polynucleotides of the invention and vectors comprising these moleculescan be used for the transformation of a suitable mammalian or microbialhost cell. Transformation can be by any known method for introducingpolynucleotides into a cell host. Such methods are well known of the manskilled in the art and include dextran-mediated transformation, calciumphosphate precipitation, polybrene-mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide intoliposomes, biolistic injection and direct microinjection of DNA intonuclei.

The humanized and engineered variants of the invention are fullyfunctional to antagonize the GPVI pathway activities. In particular,they are able to inhibit the binding of collagen to GPVI. They are alsoable to inhibit platelet aggregation induced by collagen both in humanplatelet rich plasma and in human whole blood. Further, they are alsoable to inhibit thrombus formation under flow on a collagen coatedsurface.

The binding of anti-GPVI Fab fragments of the invention to GPVI ischaracterized by the development of a GPVI depletion phenotype on theplatelet surface. This is a new and unexpected mechanism of action foranti-GPVI Fab fragments. This unique characteristic of the Fab fragmentsdescribed here, may be determined by the new epitope targeted by thesemolecules.

As used herein, “GPVI depletion phenotype” refers to the cellular statethat results when an antibody or an antigen binding fragment binds toGPVI receptor molecules on the surface of a platelet, it prevents, theactivation of the GPVI pathway on platelets. This GPVI depletionphenotype may rely on two different mechanisms: (i) either GPVIreceptors are removed or depleted from the cell surface, or (ii) theantibody or an antigen binding fragment binds to GPVI receptor moleculein a non reversible way so that inhibition of the receptor is maintainedfor the life span of the cell. The phenotype will in both situationsrevert only with the renewal of platelets.

The fact that these anti-GPVI Fab fragments induce a GPVI depletionphenotype presents two advantages in term of efficacy:

i) If receptor shedding is—at least part of the mechanism, the processis generally independent of Fab binding to GPVI. This means ifoccupation of a fraction of cell surface GPVI by the Fab (e.g. 30%) willinduce shedding, the shedding process will not be restricted to the 30%of GPVI receptors occupied by the Fab but also extended to free GPVI.This implies that one can achieve 100% of GPVI blockage (by depletion)with significantly less Fab.

ii) The inhibition effect has a long term effect: It is known that Fabfragments have a very short plasma half life of approximatively 1-2hours, which may be problematic for several indications where a longlasting inhibition of the target is required. With the describedmechanism of depletion or non-reversible occupancy of the receptors, theduration of the effect (based on pharmacodynamics properties) will beuncoupled from the pharmacokinetics properties of the Fab. This isbecause platelets are not able to replace the affected receptors, oncedepletion or non-reversible inhibition is induced Then the receptorsremain absent or unavailable for the life span of the platelet. Thatcorresponds to an extension of the duration of action to several days,depending on the half life of platelets (10 days for human platelets).

All these properties demonstrate that Fab of the present invention aresuitable candidates to the treatment of thrombotic and vasculardiseases.

Important target classes for antibody based biologics are membranereceptors that can be blocked with high specificity by monoclonalantibodies. Full length antibodies in the IgG format bind and also blocktheir target through their Fv part. The Fc part add furtherfunctionality to these molecules that lead to antibody derived cellularcytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Theseactivities are often an important part of the mAb mode of action (MoA)especially for oncology applications. However, in other applicationareas ADCC/CDC activities need to be minimized or are not necessary.Therefore, a number of antibody fragments lacking the Fc part such asFab molecules are desireable.

Another reason for developing monovalent antibody fragments derives fromthe target biology. Many membrane receptors initiate signaling afterreceptor clustering (e.g. GPVI). If such a receptor should be blocked, amonovalent antibody fragment is the natural format of choice.

Antibody fragments such as Fab fragments are much more suitable formatsthan whole antibodies for many clinical applications. This advantagerelies on several unique characteristic of Fab fragments whichdifferentiate them from whole antibodies, in particular:

-   -   monovalancy: required if cross-linking of molecules should be        avoided    -   lack of Fc domain: required if Fc-functions are not needed or        undesireable    -   lower molecular weight: determines pharmacokinetic and        pharmacodynamic behavior as well as tissue distribution        In order to maintain these desired properties of antibody Fab        fragments following the administration to a patient, interaction        of Fab fragments with non-target molecules should be avoided.

As previously mentioned, the Fab fragments are obtained by deletion ofthe constant domain of the heavy chain (Fc part) of IgG molecule.Unfortunately, removal of the Fc exposes a novel C-terminal (orC-terminal) peptide, i.e. a neoepitope. A specific class of proteins,able to specifically interact with the C-terminal extremity of Fabfragments has been reported in humans and have been identified aspreexisting antibodies against antibody fragments such as Fab (Kormeieret al. (1968) J. Immunol. 100(3);612-21; Persselin and Stevens (1985) J.Clin. Invest. 76; 723-30). These preexisting antibodies are being formedearly in life and are patient dependant. With the development ofmonoclonal antibodies and antibody fragments as novel therapeutics, theexistence of preexisting anti-Fab antibodies restricts and complicatesthe usage of therapeutic Fab molecules for the reasons mentioned above.

In addition, the C-terminal extremity of the Fab is a preferred epitopefor generation of antibodies as previously described (Christopoulos C.,et al 1994). However, even in cases were the patient has no or lowamounts of pre-existing antibodies against the C-terminus, thegeneration of new antibodies directed toward this epitope may appearafter therapeutic Fab administration, leading to this same limitationsas described with preexisting antibodies.

Indeed, binding of therapeutic Fab molecule by preexisting or newantibodies against Fab C-terminal epitope can change the pharmacokineticand pharmacodynamic behavior of the molecules (e.g. receptor activationinstead of inhibition based on the change from a monovalent to bivalentmolecules), create new complexes and functions (e.g. adding an antibodyFc-part with all its effector functions) and changes the size of thecomplex which may also have consequences for tissue distribution.Therefore, this phenomenon represents a significant safety and efficacyrisk which needs to be avoided.

In order to avoid such limitations, the neoepitope that is recognized byanti-Fab antibodies can be masked by addition of a molecule at theC-terminal end of the Fab as presently described for anti-GPVI Fabmolecules. This principle is essential for development of alltherapeutic antibody fragments in which the target biology prevents theuse of bivalent molecules (e.g. in case of receptor activation throughclustering) and where it is therefore strictly required to employmono-valent molecules.

Therefore, in order to enable the safest treatment for all patients aswell as to avoid patient specific pharmacokinetic and pharmacodynamicvariability, Fab molecules should be modified to mask Fab specificneoepitopes created at the C-terminal extremity that could be recognizedby preexisting or newly generated antibodies against Fab fragments.

This invention concerns a Fab fragment where a molecule has been addedto the C-terminus. This molecule can be a peptide or any other kind ofmolecule able to mask the epitope but without interfering with thebinding of the Fab to the target.

In a particular embodiement, the said molecule able to mask theneoepitope of the Fab fragment is a peptide which can comprise 1 to 100amino acids, 1 to 50 amino acids, 1 to 20 amino acids, 1 to 15 aminoacids or 1 to 10 amino acids.

For example, a His-tag, a G4S or (G4S)2 stretches peptide willconstitute an appropriate molecule.

In another aspect, the invention concerns a Fab bearing a molecule atthe C-terminal extremity of its heavy chain.

In particular embodiment, the Fab fragment bearing a molecule at itsC-terminal extremity specifically recognizes GPVI. In a preferedembodiement, this Fab fragment is chosen among those previouslydescribed.

In a particular embodiement, the sequence of the Fab heavy chaincorresponds to a sequence comprising SEQ ID NO. 29 or SEQ ID NO.30 orSEQ ID NO.31.

In another enbodiement, the sequence of the Fab heavy chain correspondsto a sequence consisting in SEQ ID NO. 29 or SEQ ID NO.30 or SEQ IDNO.31.

This invention also concerns a method to prevent recognition of Fabfragments by preexisting antibodies or new antibodies directed towardthe C-terminal extremity consisting in masking the C-terminal end byaddition of a molecule. As such, this molecule allows prevention ofunwanted generation of Fab-antibody complexes.

This invention is also directed toward a method of prevention ofplatelet activation when an anti-GPVI Fab is used consisting in maskingthe C-terminal extremity of the Fab by addition of a molecule.

In another aspect, the invention concerns a method for preparation of amodified Fab bearing a molecule at its C-terminal extremity comprisingthe steps of:

-   -   a. Addition of a molecule to the C-terminal extremity of the Fab    -   b. Production of the modified Fab in an appropriate system,        including in bacteria, yeast or mammalian cell lines    -   c. Purification of the modified Fab

The produced Fab can further be formulated in an appropriate solution.

In another aspect, the inventions consists in the use of an anti-GPVIantibody, in particular Fab fragments as described in the presentdescription, to prevent thrombotic events in the treatment of certainclinical indications, as for example acute coronary syndrome,percutaneous coronary intervention, ischemic stroke, carotid arterystenosis or peripheral arterial occlusive disease. Furthermore it couldbe used for the prevention of restenosis and atherosclerosis.

The invention concerns also a composition containing an anti-GPVIantibody of the invention, and in particular Fab fragments withappropriate excipients. This composition can be used to treat thromboticand vascular diseases.

In another aspect, the invention concerns a method of manufacture of anantibody according to the invention

In another aspect of the invention, the antibodies can be used fordiagnosis of GPVI expression changes. It is described that changes inthe expression of GPVI on the platelet surface as well as the occurrenceand concentration of soluble GPVI (cleaved extracellular domain of GPVI)in plasma may well be associated with pathophysiological conditions suchas acute coronary syndromes, transient ischemic attacks or stroke(Bigalke B, et al., Eur J Neurol., 2009 Jul. 21 ; Bigalke B. et al.,Semin Thromb Hemost. 2007 March;33(2):179-84).

Thus, measurement of these parameters could be used to identify patientsat risk for the aforementioned conditions requiring anti-thrombotictreatment and being possibly particularly susceptible for anti-GPVItreatment. Therefore, antibodies and antibody fragments described herecan be used as diagnostic tool and be part of a diagnostic kit whichdetermines the presence and quantitative changes of GPVI on the plateletsurface as well as in plasma samples.

The antibodies and fragments thereof can be used to diagnosis patientsat risk who could benefit of an anti-thrombotic treatment.

Such method for diagnosing of GPVI changes in a patient, may comprise(i) contacting platelets or plasma sample of said patient with anantibody or Fab fragments thereof according to the invention, (ii)measuring the binding of said antibody or Fab to the cells present insaid sample, and (iii) comparing the binding measured in step (ii) withthat of a normal reference subject or standard.

The invention also encompasses a diagnosis kit for the detection ofchanges in the human GPVI expression including an antibody of theinvention or a fragment thereof. In a particular embodiment, a kitaccording to the invention can be provided as an ELISA assay kit.

In another aspect of the invention, patients are screened for thepresence of anti-Fab antibodies prior to administration of either amasked Fab or other antibody of the invention.

The invention also provides a method for the preparation of anti-GPVIantibody or Fab fragments of the invention comprising the steps of:

-   -   a. Culture of a cell line containing DNA sequences encoding one        HC or HC fragment and one LC of the invention    -   b. Purification of the antibody or Fab expressed in the culture        medium    -   c. Formulation of the antibody in a convenient form

Any expression cell line able to produce immonuglobulin can be used inorder to express the antibodies of the invention. Expression cell linesderived from mammalian can be used as well as any other expressionsystem as for example yeast cells (A. H. Horwitz, et al PNAS. 1988November; 85(22): 8678-82) or bacterial cells (Chiba Y, Jigami Y. CurrOpin Chem Biol. 2007 December; 11(6): 670-6).

The purification of the antibody can be realized by any method known bythe person skilled in the art as for example.

The formulation of the antibody depends on the intended use of suchantibody. Depending on the use, for example pharmaceutical use,veterinary or diagnosis use, it can be lyophilized or not, besolubilized in an appropriate solution.

It should be noticed that this general description as well as thefollowing detailed description are exemplary and explanatory only andshould not be restrictive on the invention. The drawings included in thedescription illustrate several embodiments of the invention intended toexplain the principles of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Inhibition of collagen binding by competition ELISA. (A)Dose-dependency (IC50) against hGPVI-Fc and (B) against Maccaca GPVI-Fc

FIG. 2: Cross-reactivity analysis of selected hybridoma mAb againsthuman and primate GPVI by ELISA

FIG. 3: Fab produced by Ficin cleavage of IgG from hybridoma clone 390

FIG. 4: Inhibition of collagen induced platelet aggregation by anti-GPVIFab fragments (VL1 with VH3).

FIG. 5: Crystallographic data showing the interaction between the Faband the extracellular domain of the human GPVI protein. (A) Interactionbetween D2 domain of GPVI protein and the Fab. (B) Interaction betweenD1 domain of GPVI protein and the Fab. (C) Interaction between D1-D2domain of GPVI protein and the Fab.

FIG. 6: Examples of IC50 curves for the inhibitory activity of anti-GPVIFab-fragments in collagen induced whole blood aggregation.

FIG. 7: Examples of inhibition of thrombus formation under flow by antiGPVI-Fab fragments.

FIG. 8: Anti-GPVI Fab causes a reduction of GPVI surface expression in aconcentration dependent manner.

FIG. 9: Anti-GPVI Fab causes a significant GPVI depletion in a timedependent manner.

FIG. 10: Effect of anti-GPVI Fab on ex vivo whole blood plateletaggregation (agonist : 1 μg/ml). A—after 1 mg/kg IV bolusadministration. B—after 3 mg/kg IV bolus administration.

FIG. 11: In vitro activity of anti-GPVI Fab in whole blood plateletaggregation assay (agonist: 1 μg/ml)

FIG. 12: Effect IV bolus administration of anti-GPVI Fab on plateletcount. A—After PBS vehicule at 3ml/kg (control). B—After 0.01 mg/kg ofanti-GPVI FAb. C—After 0.1 mg/kg of anti-GPVI FAb. D—After 1 mg/kg ofanti-GPVI FAb. E—After 3 mg/kg of anti-GPVI FAb.

FIG. 13: FACS analysis of GPVI receptor expression on platelets after ivadministration of anti-GPVI Fab. The vertical line descriminates betweenGPVI negatives platelets on the left side and GPVI positive platelets onthe right side. A—pre-dose. B—at 24 h after administration. C—at 48 hafter administration. D—at 72 h after administration. E—at 150 h afteradministration.

FIG. 14: Effect of anti-GPVI Fab on platelet activation. In controlpanel, addition of convulxin. In panel A, addition of Fab after IgGdepletion. In panel B, addition of modified Fab.

FIG. 15: A—Fab control; B—Fab-His-Tag ; C—FabGly4Ser and Fab-(Gly4Ser)2

FIG. 16: GPVI with the 20 residues signal sequence removed (correspondsto SEQ ID NO: 32). Bolded residues represent the conformational epitopemaking contact with antibody CDRs

EXAMPLES Example 1 Generation of Recombinant Extracellular D1 and D2Domain of GPVI

A—Construction of hGPVI-hFc Fusion Expression Plasmid (hGPVI-hFc)

Using human cDNA containing plasmids as a PCR template, a DNA fragmentencoding a 237 amino acid residue heavy chain constant region includingthe hinge region, CH2 and CH3 domains of human immunoglobulin IgG wasamplified.

Using human genomic DNA as PCR template, a DNA fragment encoding a 205amino acid residue human GPVI D1 and D2 extracellular domains wasamplified. This human GPVI D1 and D2 fragment includes the signalsequence and corresponds to amino acids M1 to T205 in the wild typeprotein (NP_(—)057447/Q9HCN6). The resulting amplified, cleaved andpurified PCR products encoding human GPVI D1 and D2 and a human Fcregion were combined by ligation PCR and ligated into baculovirusexpression vector pVL1393 by InFusion method using EcoRI and NotI site.The resulting GPVI-Fc ORF is listed as SEQ ID NO.1 and its correspondingprotein sequence as SEQ ID NO.2.

B—Construction of GPVI-tev-his Expression Plasmid (GPVI-tev-his)

Using the previously described GPVI-Fc containing plasmid as PCRtemplate, human GPVI D1 and D2 extracellular domains were amplified,including the signal sequence corresponding to amino acids M1 to T205 inthe wild type protein (Swissprot: Q9HCN6). The reverse primer containedDNA coding for a tev cleavage recognition sequence and 7 histidineresidues representing the His-tag at the C-terminus of the construct.The resulting amplified PCR fragment was cleaved with the restrictionendonucleases EcoRI+NotI and was ligated into baculovirus expressionvector pVL1393. The resulting ORF is listed as SEQ ID NO.3 and itscorresponding protein sequence as SEQ ID NO.4.

C—Expression and Purification of hGPVI-hFc and GPVI-tev-his Protein

SF9 cells growing in SF900 II serum free suspension culture (Invitrogen)were cotransfected with the expression plasmid and FlashBac baculovirusDNA (Oxford Expression Technologies). Transfection was performed usingCellfectin transfection reagent (Invitrogen). After 5 h, 5% total bovinefetal serum was added to the transfected culture. The cells werecultured at 28° C. for 5 days. The culture supernatant containingrecombinant virus was used for larger scale virus amplification in SF900II suspension culture containing 5% bovine fetal serum.

For protein expression HighFive (Invitrogen) cells growing in ExCell 405(SAFC) were transduced with an appropriate amount of virus stock andgrown at 27° C. for 72 h. After harvest the cell culture supernatant wasclarified by filtrated (0.22 μm).

For purification the Fc-fusion GPVI protein variants were captured onprotein A matrix (GE Healthcare) and eluted by pH shift. After polishingthe protein by SEC using a Superdex 200 (GE Healthcare) and a finalultrafiltration concentration step the protein was used for ELISA andfuther assays.

For purification the His-tagged GPVI protein variants were directlycaptured from supernatant on IMAC matrix (HisTrap, GE Healthcare) andeluted by an imidazol gradient. After polishing the protein by SEC usinga Superdex 75 (GE Healthcare) and ultrafiltration the protein was usedin indicated experiments.

Example 2

Generation and Selection of Functional anti-GPVI mAbs

A—Generation of anti-GPVI mAbs

GPVI-specific antibodies were generated using the RIMMS method asdescribed by Kilpatrick et al. (1997. Hybridoma 16: 381389).

6-8 weeks old female BALB/c mice (S082342; Charles River Labs, BarHarbor, Me.) each received four rounds of immunization with purifiedsoluble his-tagged GPVI protein (prepared as described in Example 1)over a course of 14 days at intervals of 3-4 days.

For the first immunization on day zero, 5 μg antigen emulsified inTitermax's adjuvant (TierMax Gold Adjuvant; Sigma #T2684) wasadministered subcutaneously to six sites proximal to draining lymphnodes, along the back of the mice. Another 5 μg of antigen emulsified inRIBI's adjuvant (Sigma Adjuvant system; Sigma #S6322) was administeredto six juxtaposed sites along abdomen. Booster immunizations were givenon days 4, 7 and 11 in a similar fashion.

Four days after the last injection, mice were sacrified. Bilateralpopliteal, superficial inguinal, axillary and branchial lymph nodes wereisolated aseptically and washed with fresh RPMI medium. Lymphocytes werereleased from the lymph nodes and the resulting single-cell suspensionwas washed twice with RPMI medium before being fused with P3X63-AG8.653myeloma cells using polyethylene glycol. After fusion, the cell mixturewas incubated in an incubator at 37° C. for 16-24 hours. The resultingcells preparation was transferred into selective semi-solid medium andaseptically plated out into 100 mm Petri plates and incubated at 37° C.

Ten days after initiation of selection, the plates were examined forhybridoma growth, and visible colonies were picked-up and placed into96-well plates containing 200 μL of growth medium. The 96-well plateswere kept in an incubator at 37° C. for 2 to 4 days.

B—Screening for mAbs Recognizing the Human GPVI Protein

Primary screening for anti-GPVI IgG production was performed by ELISAusing GPVI-Fc fusion protein (prepared as described in Example 1) ascapturing antigen. Plates were coated with GPVI-Fc fusion protein andhybridoma supernatants were added to the plate and detected by usingrabbit anti-mouse IgG conjugated with horseradish peroxidase (Sigma;#A9044). Antibody binding was visualized by adding TMB-H2O2 buffer andread at a wavelength of 450 nm.

Among 367 hybridomas selected from 96-well plates, 129 hybridomas werepositive for anti-human GPVI antibody production and then 111 wereconfirmed after cell amplification.

C—Ability of anti-GPVI mAbs to Block Binding of Collagen to Human GPVI

A secondary screening was performed to characterize functionalproperties of all human GPVI-specific mAbs for their ability to blockthe binding of collagen to human GPVI-Fc fusion protein in a competitionELISA binding assay. Custom-made collagen coated 96-well plates (Pierce)were used. Pre-incubated mixture of human GPVI-Fc fusion protein andhybridoma supernatants were added to the plate and collagen-humanGPVI-Fc complex was detected by using goat anti-human IgG-Fc conjugatedwith horseradish peroxidase (Sigma; #A0170). The antibody binding wasvisualized by adding TMB-H2O2 buffer and read at a wavelength of 450 nm.Among the 100 GPVI-binding hybridomas, 22 hybridomas blocked the bindingof GPVI-Fc to collagen (threshold: 90% inhibition). The blockingproperties of pre-selected mAbs to human GPVI were confirmed to bedose-dependent using a competition ELISA assay as shown in FIG. 1A.

All antagonist mAbs were isotyped as IgG1, kappa, as determined by usinga mouse IgG isotyping kit (SEROTEC; #MMT1) (data not shown).

D—Binding Properties of the anti-GPVI mAbs

A last screening was performed by Surface Plasmon Resonance (BIAcore2000) to evaluate binding properties of GPVI blocking antibodies. Inthis analysis, we evaluated the interaction of the human GPVI proteinwith the anti-human GPVI mAbs fixed to anti-Fc antibody covalentlylinked to CM chips. Binding kinetics of the individual mAbs wereperformed using the protocol described by Canziani et al 2004. Anal.Biochem. 325 : 301-307.

All blocking mAbs displayed affinities in the (sub)nanomolar range tohuman GPVI (Table 1). Based on target affinities, the antibody fromhybridoma clone 390 was selected for further development.

TABLE 1 Affinity and association/dissociation rates of selectedanti-hGPVI mAbs Biacore Hybridoma ka (1/Ms) kd (1/s) K_(D) (M) 256.05E+04 2.57E−04  4.2E−09 29 15.4E+04 3.28E−04  2.1E−09 136 15.2E+043.16E−04  2.0E−09 145 5.87E+04 8.63E−04 14.7E−09 149 1.27E+04 1.49E−0411.7E−09 202 1.33E+04 2.10E−04 15.8E−09 298   85E+04 18.2E−04  2.1E−09345 16.7E+04 3.29E−04  1.9E−09 390 10.8E+04 0.04E−04 0.04E−09

E—Cross-Reactivity Properties of the anti-GPVI mAbs with the PrimateGPVI Protein

GPVI-specific mAbs listed in table 1 were assessed for their ability tobind primate GPVI-Fc protein by ELISA. Plates were coated with primateGPVI-Fc fusion protein, anti-hGPVI mAbs were added to the plate anddetected with rabbit anti-mouse IgG conjugated with horseradishperoxidase (Sigma; #A9044). The antibody binding was visualized byadding TMB-H2O2 buffer and read at a wavelength of 450 nm. Thepre-selected hybridomas were cloned by growth in semi-solid medium.Petri plates were seeded at 125 cell/mL and clones showing significantgrowth were screened for GPVI binding, GPVI blocking activity and crossreactivity with primate GPVI. As shown in FIG. 1B and FIG. 2, all mAbswere cross-reactive with primate GPVI.

The sequences used for extracellular domain of primate (Macacafascicularis or cynomolgus) GPVI are described as SEQ ID NO.27 and SEQID NO.28.

F—Determination of the Sequence of the Heavy and Light Chains of theanti-GPVI mAbs

The cDNA encoding the variable domains of the monoclonal antibodies wereobtained as follows: mRNA was extracted from hybridoma cells with theOligotex kit from Qiagen. The corresponding cDNA was amplified by RT-PCRby the RACE method utilizing the Gene Racer kit (Invitrogen), thetranscriptase SuperScript III at 55° C. (Invitrogen) and primersdescribed on Table 2 (RACEMOG1 or CKFOR). The cDNA fragments wereamplified by PCR with the polymerase Phusion at 55° C. (Finnzymes) andprimers also described in Table 2.

TABLE 2 Primers used for RT-PCR and PCR Primer Sequence number5′-GeneRacer Primer SEQ ID NO. 24 RACEMOG1: 3′-Primer internal SEQ IDNO. 25 to murin hinge murin CKFOR: 3′-Primer internal to SEQ ID NO. 26murin Ck murin

The amplified fragments encoding the variable regions of heavy (VH) andlight (VL) chains were cloned into the pGEM-T Easy plasmid from Promegaor pCR4-Topo plasmid from Invitrogen which were amplified in E. coli.Cloned cDNA was then sequenced on both strands.

Protein sequences were translated from plasmid coding sequences and themasses of the heavy (HC) and light (LC) chains were calculated (Table3). The values obtained were in perfect agreement with mass spectrometrydata obtained from preparations of mAbs purified from cultures of thecorresponding hybridoma, see Table 3. In particular, the occupancy of aN-glycosylation site in the variable region of the heavy chain (NST) ofanti-GPVI antibody 390 was confirmed. Amino acid sequence of HC and LCare reported in the sequence listing as follows: SEQ ID NO.5 and SEQ IDNO. 6 corresponds to the HC of anti-GPVI mAb from clone 390 and SEQ IDNO.7 and SEQ ID NO. 8 correspond to the LC of anti-GPVI mAb from clone390.

TABLE 3 Mass spectrometry analysis of Anti-GPVI mAbs from hybridomaAnti-GPVI Mass (Da) Mass (Da) mAb Chain by LC/MS in silico value 390 LC23417 23414 HC 51127 49586 (G0)* *Compatible with additional highmannose N-glycan. Mass of 49586 Da confirmed by LC/MS analysis afterdeglycosylation.

G—Determination of the Sequences of the CDR of the anti-GPVI mAbs

The sequences for the CDR regions were deduced from the protein sequenceusing the KABAT nomenclature.

For the HC, CDR1 corresponds to SEQ ID NO.18, CDR2 corresponds to SEQ IDNO.19 CDR3 corresponds to SEQ ID NO.20

For the LC, CDR1 corresponds to SEQ ID NO.21, CDR2 corresponds to SEQ IDNO.22 CDR3 corresponds to SEQ ID NO.23

Example 3

Preparation and Biophysical Properties of anti-GPVI mAbs and their FabFragments

A—Inhibition of Collagen Binding to Recombinant GPVI by anti-GPVI IgG'sor their Proteolytic Fab Fragments

Collagen coated 384well plates (Pierce) were blocked with 3% BSA for 2h. Increasing concentrations of IgG or its corresponding Fab fragmentproduced by Ficin cleavage (0,3-20 μg/ml) were incubated withrecombinant GPVI (Fc-fusion protein of the extracellular GPVI domain; 3μg/ml) for 30 min. The GPVI-IgG mixture was added to collagen coatedplates and incubated for 1 h at RT. Next, 384well plates were washed(DELFIA wash buffer, Perkin Elmer) five times and Eu-labelled anti-humanIgG (100 ng/ml Perkin Elmer) was added. Following 1 h incubation at roomtemperature plates were washed again five times, enhancement solution(Perkin Elmer) was added and incubated for 10 min. Fluorescence wasdetected at 360/612 nm using a Tecan Ultra reader. FIG. 3 shows atypical readout. Table 4 shows calculated IC50 values (μg/ml) forinhibition of collagen binding to GPVI of two independent experiments.

TABLE 4 Calculated IC50 values (μg/ml) for inhibition of collagenbinding to GPVI of two independent experiments. Hybridoma IgG Fab (Ficincleavage) Clone ID Test 1 Test 2 Test 1 Test 2 390 5.6 5.0 1.3 1.1

B—Production of Recombinant Fab Fragments

1. Construction of Expression Plasmids for Recombinant Production ofanti-GPVI Fab

Amino acid sequences of the variable heavy and light chains of theanti-human GPVI antibodies were backtranslated into nucleotide sequenceand generated respectively using either a modified protocol of theoverlap extension PCR (OE-PCR) described by Young L. and Dong Q. (Nucl.Acids Res. (2004), 32(7), e59) or by gene synthesis (Geneart). PCRproducts were cloned into the pCR Blunt II TOPO vector using theInvitrogen TOPO cloning kit and sequenced using M13forward and M13reverse primers. Each variable heavy chain was fused to the CH1 domainof IGHG1 (Genebank accession number Q569F4) and the variable light chainwas fused to the constant kappa chain (IGKC, Genebank accession numberQ502W4) respectively.

These fragments were digested with NheI and HindIII and each ligatedinto the NheI/HindIII sites of the episomal expression vector pXL, ananalogon of the pTT vector described by Durocher et al. (2002), Nucl.Acids Res. 30(2), E9, creating the plasmids for transient mammalianexpression of the chimeric and humanized anti-GPVI Fabs. The expressionplasmids encoding the heavy and light chain of the antibody werepropagated in E. coli NEB 10-beta (DH10B derivative). Plasmids used fortransfection were prepared from E. coli using the Qiagen EndoFreePlasmid Mega Kit.

2. Transient Expression and Purification of Recombinant anti-GPVI FabFragments

Hek 293-FS cells growing in Freestyle Medium (Invitrogen) weretransfected with indicated LC and HC plasmids using Fugene (Roche)transfection reagent. After 7 days the cells were removed bycentrifugation, 10% Vol/Vol 1M Tris HCl pH 8,0 was added and thesupernatant was passed over a 0.22 μm filter to remove particles. TheFab proteins were captured using KappaSelect matrix (GE Healthcare) andeluted via pH shift. The protein containing fractions were pooled anddesalted using PD-10 or Sephadex columns. Concentrated and sterilefiltered (0.22 μm) protein solutions were adjusted to 1 mg/ml and keptat 4° C. until use.

C—Biophysical Characterization of Recombinant Fab

Surface plasmon resonance technology on a Biacore 3000 (GE Healthcare)was used for detailed kinetic characterization of purified antibodyfragments. A direct binding assay was used with the anti-GPVI Fab as theligand and human GPVI as analyte. Typically, 500 RU of anti-GPVI Fabwere immobilized on a research grade CM5 chip by amine reactivecoupling, resulting in an Rmax of 200 RU for the bound GPVI molecule.Binding kinetics were measured over a concentration range typicallybetween 0.8 to 208 nM in HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mMEDTA, 0.005% Surfactant P20) at a flow rate of 30 μl/min. Chip surfaceswere regenerated with 10 mM glycine pH 2.0. Kinetic parameters wereanalyzed and calculated in the BIAevaluation program package (version4.1) using a flow cell without immobilized Fab as reference. A 1:1binding model with mass transfer was applied for a global fit of thedata for curves.

In order to investigate functional consequences of the additionalglycosylation motif present in the original sequence as derived fromglycosylated clone 390 (Fab 390-G), the conserved glycosylation motifwas removed by amino acid replacement (Fab 390-nG). For this purpose,two heavy chain fragments based on clone 390 were constructed: the firstone does not have any modification and corresponds to SEQ ID NO.9, andthe second one has a point mutation N→Q at position 60 of SEQ ID NO.9.

It is known that glycosylation can be very heterogeneous and produceglycoprotein heterogeneity; such source of heterogeneity should beavoided as much as possible.

In the present case, the removal of the additional glycan does notimpact the binding affinity for GPVI as shown in Table 5. Thus, thenon-glycosylated Fab 390-nG construct has been chosen for furtherexperiment.

TABLE 5 Determination of binding characteristics for recombinantanti-GPVI Fab molecules by SPR (Biacore) Recombinant Fab ka (1/Ms) kd(1/s) KD (M) Construct E+04 E−04 E−09 Fab 390-G 32 0.15 0.05 Fab 390-nG34.8 0.22 0.06

D—Determination of the Epitope Recognized by the anti-GPVI mAb byCo-Crystallization of anti-GPVI Fab with GPVI and X-ray Crystallography

For epitope characterization a co-crystallization strategy has beenapplied. A complex between Fab 390-nG (expressed in HEK293 cells) andhuman glycoprotein VI (extracellular domain of human GPVI (Met1-Thr205),expressed in Insect cells High five) was formed by incubation of bothproteins at equal molar ratio. The complex was digested with trypsin,re-purified by gelfiltration, concentrated and subjected tocrystallization screening. Well diffracting crystals were obtained bymixing 100 nl of protein solution (Fab-antigen complex at 6.5 mg/ml in20 mM Tris pH 8.0, 150 mM NaCl) with 100 nl of reservoir solutioncontaining 0.1M BisTris pH 5.5, 25% PEG3350 and 0.2M ammonium acetateand incubating the protein drop against 200 μl reservoir solution at 4°C. in a sealed sitting drop vapour diffusion crystallization plate. Onecrystal was flash frozen in liquid nitrogen using reservoir solutionsupplemented with 25% ethylene glycol for cryoprotection.Crystallographic data was collected at the European SynchrotronRadiation Facility, Grenoble, France, at beamline ID14-4. The structurewas solved by Molecular Replacement using the Fab fragment of thepdb-entry 1FDL as model for Fab 390-nG and one monomer of the crystalstructure of human platelet glycoprotein VI, pdb-entry 2G17, as modelfor GPVI. The resolution and final R-factor of the refined structure are1.72 Å, R-factor 17.2% and R-free 19.9%. The GPVI-binding epitope is aconformational epitope characterized by interactions (defined asdistance<4.5 Å) of Fab 390-nG to the following residues of the antigenhGPVI:

Pro4, Lys7, Glu40, Lys41, Leu42, Ser43, Ser44, Ser45, Arg46, Arg65,Arg67, Trp76, Leu78, Pro79, Ser80, Asp81, Gln82, Ser165, Arg166

These residues belong either to the D1 domain (up to amino acid 84) orconstitute parts of the D2 domain (amino acids 94 to 176) and thereforerepresenting a conformational epitope. The numbering system for GPVIepitope residues corresponds to the protein with the signal sequenceremoved as shown in FIG. 16 and SEQ ID NO: 32. The interaction of Fabanti GPVI from clone 390 with the extracellar D1-D2 domain of GPVI isillustrated on FIG. 5.

The antibody residues contacting GPVI epitope residues are described inTables 6 through Table 11 below.

As used herein, “contacting residues” were defined as antibody residueswith a distance of less than 4.5 Å to GPVI antigen residues or thereverse and as measured in the crystal structure between antibody toGPVI and GPVI antigen. The distances were determined using the softwareprogram NCONT of the CCP4 Software package.

The X-ray structure highlights residues from the CDRs that can bemutated, that should not impact binding to GPVI.

In the following descriptions, residues from the CDRs might be modifiedas indicated, without disruption of the binding to GPVI. It is alsounderstood that mutations in the CDRs should not affect the conformationand orientation of the residues from the CDRs to preserve binding toGPVI. Residue numbering is sequential and does not follow Kabbatconventions as used in Al-Lazikani ((1997) J. Mol. Biol. 273, 927-948).“X” represents “any residue”, while “−” indicates that no modificationshould be made at this position.

CDRH1 sequence as defined in SEQ ID NO: 18 spans ten residues. As shownin Table 6, six of ten or 60% of CDRH1 residues do not make contact withGPVI. All of the GPVI residues in contact with antibody residues areserine residues.

TABLE 6 Antibody CDRH1 residues contacting GPVI antigen residues H26 H27H28 H29 H30 H31 H32 H33 H34 H35 CDRH1 G F S L T G Y G V N Initialsequence Suggested — — X — X X Y/F — — — mutations GPVI S44 S43 S43 S43S44 S44 S44

CDRH2 sequence is defined in SEQ ID NO: 19 and spans sixteen antibodyresidues. As shown in Table 7, nine of sixteen or 56% of CDRH2 residuesdo not make contact with GPVI. Tryptophan 52 was found to be in contactwith seven different GPVI antigen residues. Arginine 46 of GPVI wasfound in contact with five different CDRH2 residues.

TABLE 7 Antibody CDRH2 residues contacting GPVI antigen residues 50 5152 53 54 55 56 57 58 59 60 61 62 63 64 65 CDRH2 M I W G D G S T D Y Q ST L K S Initial sequence Suggested — I/V/L — — — — S/T X — Y/F — X X L/IX X mutations GPVI S43 E40 S43 S44 R46 R46 R46 K41 S44 S45 L42 S45 R46S43 S44 S45 R46

CDRH3 sequence is defined in SEQ ID NO: 20 and spans five antibodyresidues. Based on the fact the definition of the CDR sequence canslightly vary depending on the software used, it has been found thatCDRH3 may include one additional residue. Thus CDRH3 can be as definedin SEQ ID NO:38, spanning six antibody residues. As shown in Table 8 twoof six or 33% of CDRH3 residues do not make contact with GPVI. Arginine65 was found to be in contact with three different GPVI antigenresidues. There are three different arginine residues of GPVI contactingCDRH3 residues.

TABLE 8 Antibody CDRH3 residues contacting GPVI antigen residues 98 99100 101 102 103 CDRH3 D L P M D Y Initial sequence Suggested D/N L/I/V —— D/N X mutations GPVI L42 L42 R65 R166 S43 S43 R65 R65 R67

CDRL1 sequence is defined in SEQ ID NO: 21 and spans eleven antibodyresidues. As shown in Table 9, seven of eleven or 64% of CDRL1 residuesdo not make contact with GPVI. Tyrosine 32 was found to be in contactwith five different GPVI residues.

TABLE 9 Antibody CDRL1 residues contacting GPVI antigen residues 24 2526 27 28 29 30 31 32 33 34 CDRL1 K A S Q D I N K Y I A Initial sequenceSuggested X — X G/E D/N — — X — — — mutations GPVI P4 L78 K7 R67 P79 P79S80 S80 D81 D81 Q82

CDRL2 sequence is defined in SEQ ID NO: 22 and spans sevenantibodyresidues. Based on the fact the definition of the CDR sequence canslightly vary depending on the software used, it has been found thatCDRL2 may include one additional residue. Thus CDRL2 can be as definedin SEQ ID NO: 39 spanning eight antibody residues. As shown in Table 10,six of eight or 75% of CDRL2 residues do not make contact with GPVI.This CDR is in contact with residues in both D1 and D2 domains of GPVI.Tyrosine 50 is in contact with asp 81 and gln 82 in D1, while proline 56is in contact with arg 166 and ser 165 of GPVI D2 domain.

TABLE 10 Antibody CDRL2 residues contacting GPVI antigen residues 50 5152 53 54 55 56 57 CDRL2 Y T S T L Q P G Initial sequence Suggested — T/SX X X X — X mutations GPVI D81 S165 Q82 R166

CDRL3 sequence is defined in SEQ ID NO: 23 and spans eight antibodyresidues. As shown in Table 11, five of eight or 63% of CDRL3 residuesdo not make contact with GPVI.

TABLE 11 Antibody CDRL3 residues contacting GPVI antigen residues 89 9091 92 93 94 95 96 CDRL3 L Q Y A N L L T Initial sequence Suggested — — —— N/D/Q/E/S/T/R/K X — T/S mutations GPVI L42 R67 W76 R67 P79

A summary of the contacts between GPVI residues interacting withantibody residues revealed that ser 43 has 8 contacts, ser 44 has 7contacts, arg 46 has 4 contacts, arg 67 has 4 contacts, arg 65 has 3contacts, asp 81 has 3 contacts, and Arg 166 has two contacts. Most GPVIresidues favor either the light chain or the heavy chain, but R67 andR166 have contact with both.

E—Humanization of the Fv Domain of anti-GPVI Fab

The humanization protocol described in the patent application WO2009/032661 was used to humanize the Fab 390-nG clone. In addition,analysis of the crystallographic complex between Fab 390-nG and thehuman GPVI led to several mutations with the aim of improving or atleast retaining the affinity of Fab 390-nG to human GPVI within thehumanization campaign.

The VL & VH sequences of Fab 390-nG were blasted against the August 2007version of the Protein Data Bank (PDB). The PDB structure 1L71 was usedto build up a homology model of the light chain. The PDB structures 1YY8and 1ZA6 were both used to build up a homology model of the heavy chain.The 1ZA6 structure was specifically used to model the CDR3 subregion,while the 1YY8 structure was used for the remaining part of the heavychain. The resulting VL and VH models were used to build up a homologymodel of the variable domains which was subsequently energy minimizedusing the standard procedure implemented in Molecular OperatingEnvironment (MOE). MOE is a software for computer assisted drug designdistributed by the Chemical Computing group. The minimized model of Fab390-nG was subsequently submitted to Molecular Dynamics simulations inorder to identify the most flexible residues which are more likely tointeract with T-cell receptors and responsible for activation of theimmune response. The simulations were run with the AMBER softwaredistributed by the University of California. 57 amino-acids are finallyidentified as flexible in the Fab 390-nG. The motion of the most 60flexible Fab 390-nG amino-acids (excluding the CDR+5 Å region), duringthe 20 ns (10×2ns), were then compared to the motion of thecorresponding flexible amino-acids of 49 human germlines homologymodels, for each of which were run the 10×2 ns MD simulations. The 49human germlines models were built by systematically combining the 7 mostcommon human germline light chains (vk1, vk2, vk3, vk4, vlambda1,vlambda2, vlambda3) and 7 most common human germline heavy chains (vh1a,vh1b, vh2, vh3, vh4, vh5, vh6). The vk1-vh6 human germline sequencesshowed a 84% 4D similarity of its flexible amino-acids compared to theflexible amino-acids of the Fab 390-nG sequences; the vk1-vh6 germlinesequences were therefore used to humanize the Fab 390-nG sequencesfocusing on the flexible amino-acids. For the pairwise amino-acidassociation between Fab 390-nG and vk1-vh6 amino-acids, the 2 sequenceswere aligned based on the optimal 3D superposition of the alpha carbonsof the 2 corresponding homology models. The following motifs ofpotentially problematic sequences were considered in some cases: Asp-Pro(acide labile bond), Asn-X-Ser/Thr (glycosylation, X=any amino-acid butPro), Asp-Gly/Ser/Thr (succinimide/iso-asp formation in flexible areas),Asn-Gly/His/Ser/Ala/Cys (exposed deamidation sites), Met (oxidation inexposed area). The resulting engineered sequences were blasted forsequence similarity against UniProtKB/Swiss-Prot database providingconfidence that reasonable assumption has been made. In addition none ofthe sequences contains any known B- or T-cell epitope listed in theImmune Epitope Database and Analysis Resource (IEDB database).

Five versions for the heavy chain (Fab 390-nG VH1, VH2, VH3, VH4, VH5)and three versions were suggested for the light chain (VL1, VL2, VL3).All versions derive from the sequences of the ANTI-GPVI Fab 390-nGconstruct. The starting sequences for unhumanized Fab 390-nG areprovided in SEQ ID NO33: for VH and SEQ ID NO:34 for VL. The L1 versionhas 4 mutations. The L2 version includes one additional mutation (N93H)to potentially improve the binding to human GPVI. The L3 version derivesfrom L1 and includes an additional mutation to potentially improve thebinding to human GPVI (N93L).

Version H1 contains 5 humanizing mutations derived from the closesthuman heavy chain germline sequence, VH6, as found following thepreviously described procedure. Version H2 contains 4 humanizingmutations and derives from H1 without mutating the amino-acid inposition 73 (Asn73), which was found to be important for a potentaffinity as seen in the crystallographic complex between Fab 390-nG andthe human GPVI. Version H3 contains 5 humanizing mutations derived fromthe human heavy chain germline sequence VH3. VH3 was found to be closeto the VH chain of Fab 390-nG, following the previously describedprocedure, and to have an asparagine (Asn) residue in position 73. TheH4 version derives from H2 and includes one additional mutation (G31Y)to potentially improve the binding to human GPVI. The H5 version derivesfrom H2 and includes one additional mutation (Y103E) to potentiallyimprove the binding to human GPVI.

Eight combinations of VL and VH variants were recommended for generationof engineered antibodies: VL1 with VH1, VL1 with VH2, VL1 with VH3, VL2with VH2, VL3 with VH2, VL1 with VH4, VL1 with VH5 and VL3 with VH5. Asshown in Table 12 and Table 13, the amino acid changes were made inengineered VL and VH variants of the Fab 390-nG, using the methodologyset forth in the detailed description section of the instantapplication. The left column indicates the original amino acids andtheir positions in the murine Fab 390-nG.

Besides the engineering of the variable domain, the antibodies and theirfragments can be further modified by the addition of tags or amino acidextensions. For example a sequence of six or more histidine residues, inparticular (His)₆, or (His)₇, or (His)₈, could be added at theC-terminus of the heavy chain or the terminus could be extended insequence by addition of amino acids such as GlyGlyGlyGlySer or(GlyGlyGlyGlySer)₂ units. Similarly the framework of the antibodies andtheir fragments could be changed from an IgG1 backbone to another IgGbackbone like IgG4.

TABLE 12 Summary of the mutations introduced into SEQ ID NO: 34 for thehumanized light chain of the anti-GPVI Fab 390-nG construct Light Chain(Sequential numbering as shown in SEQ ID NO: 34) L1 L2 L3 LEU15 VAL VALVAL LYS18 ARG ARG ARG ILE58 VAL VAL VAL GLU79 GLN GLN GLN ASN93 ASN HISLEU 4 mutations 5 mutations 5 mutations

TABLE 13 summary of the mutations introduced into SEQ ID NO: 33 for thehumanized heavy chain of the anti-GPVI Fab 390-nG construct Heavy Chain(Sequential numbering as shown in SEQ ID NO: 33) H1 H2 H3 H4 H5 GLN1 GLNGLN GLU GLN GLN LYS5 GLN GLN LEU GLN GLN GLN16 GLN GLN GLY GLN GLN GLY31GLY GLY GLY TYR GLY GLY42 SER SER GLY SER SER LYS43 ARG ARG LYS ARG ARGASN73 THR ASN ASN ASN ASN GLN86 THR THR ARG THR THR TYR103 TYR TYR TYRTYR GLU GLN106 GLN GLN LEU GLN GLN 5 4 5 5 5 mutations mutationsmutations mutations mutations

F—Biophysical Characterization of Humanized Variants

Surface plasmon resonance technology on a Biacore 3000 (GE Healthcare)was used for detailed kinetic characterisation of purified antibodyfragments. An assay with immobilised human GPVI was used. Typically, 300RU of GPVI were immobilised on a research grade CM5 chip by aminereactive coupling, resulting in an Rmax of 200 RU for the bound antibodyfragment. Binding kinetics were measured over a concentration rangebetween 3,2 to 112 nM in HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mMEDTA, 0.005% Surfactant P20) at a flow rate of 30 μl/min. Chip surfaceswere regenerated with 10 mM glycine pH2.0. Kinetic parameters wereanalysed and calculated in the BIAevaluation program package (version4.1) using a flow cell without immobilised GPVI as reference. A 1:1binding model with mass transfer was applied for a global fit of thedata for curves corresponding to analyte concentrations from 3-56 nM ofFab fragment.

The binding kinetics of anti-GPVI antibody fragments from thehumanisation campaign are shown in Table 14. Note that the Biacore assayused to measure the data in this example is different in terms ofimmobilized binding partner (GPVI versus the mAb/Fab fragment) and thisleads to the different Biacore affinities as reported in Table 5.

TABLE 14 Determination of binding characteristics of humanized variantsof Fab 390-nG against extracellular domain of human GPVI by SPR(Biacore) LC/HC- ka (1/Ms) kd (1/s) KD (M) Combination E+04 E−04 E−09Fab 390-nG 10.2 0.2 0.2 VL1/VH1 11.1 0.4 0.4 VL1/VH2 11.3 0.3 0.2VL1/VH3 14.8 0.2 0.1 VL1/VH4 12.4 0.2 0.2 VL1/VH5 8.1 0.3 0.4 VL2/VH210.8 0.3 0.3 VL3/VH2 13.2 0.3 0.2 VL3/VH4 15.1 0.4 0.3

Example 4

Inhibition of Collagen Binding and of Platelet Aggregation by anti-GPVIFab Fragments. A. Inhibition of Binding of Recombinant GPVI to Collagenby Recombinant anti-GPVI Fab Fragments Collagen coated 384well plates(Pierce) were blocked with 3% BSA for 2 h. Increasing concentrations ofanti GPVI Fab fragments (0,3-20 μg/ml) were incubated with recombinantGPVI (Fusion protein of the extracellular domain of GPVI and Fc-Part ofhuman IgG; 3 μg/ml). The GPVI-Fab mixture was added to collagen coatedplates and incubated for 1 h at RT. 384well plates were washed (DELFIAwash buffer, Perkin Elmer) five times and Eu-labelled anti-human IgG(100 ng/ml Perkin Elmer) was added. Following 1 h incubation at roomtemperature plates were washed again five times, enhancement solution(Perkin Elmer) was added and incubated for 10 min. Fluorescence wasdetected at 360/612 nm using a Tecan Ultra reader. Shown in Table 15 areexamples of the inhibitory effect of recombinantly produced anti-GPVIFab fragments on GPVI collagen binding.

TABLE 15 Measured IC50 values (μg/ml) for the inhibition of collagenbinding to GPVI of three independent experiments. Variant Test 1 Test 2Test 3 Based on 0.49 0.56 0.99 clone 390* VL1/VH1 1.10 0.75 1.00 VL1/VH21.17 0.82 — VL1/VH3 0.63 1.24 0.87 VL1/VH4 1.01 1.14 1.06 VL1/VH5 1.411.58 1.24 VL2/VH2 0.81 0.73 1.05 VL3/VH2 0.67 0.90 1.04 VL3/VH4 0.720.72 1.02 *Note: This variant corresponds to a non-humanized constructbased on the original clone 390 without the His-tag. Therefore thisconstruct is exactly in the same format as the humanized variants (DKTHTat the C-terminus of HC) mentioned in the same table.

B. Inhibition of Collagen Induced Platelet Aggregation by Recombinantanti-GPVI Fab Fragments (Human Platelet Rich Plasma)

For platelet rich plasma (PRP), blood was collected into syringescontaining ACD-A to a final concentration of 10%. After centrifugationat 200×g for 20 min at room temperature without brake, supernatant (PRP)was separated. Platelet poor plasma (PPP) was separated from theremaining blood by centrifugation for 10 min at 1500×g. PRP was set to aplatelet count of 3.0×108 cells/ml by dilution with PPP. Fab fragmentswere used at final concentration of 0.15-20 μg/ml and incubated with PRPfor 5 min at 37° C. Thereafter collagen was added at a concentration of1-1,5 μg/ml and the aggregation response was monitored by themeasurement of light transmission by either using a 96well plate reader(96well plate aggregation) or Born aggregometer (classical aggregation).The aggregation response was monitored for 20 min. As shown in Table 16all investigated Fab fragments were able to inhibit collagen inducedplatelet aggregation in a concentration dependent manner.

TABLE 16 Calculated IC50 values (μg/ml) for inhibition of collageninduced platelet aggregation of three independent experiments. VariantTest 1 Test 2 Test 3 Based on 1.6 6.4 1.3 clone 390* VL1/VH1 1.5 2.3 —VL1/VH2 2.3 2.0 1.1 VL1/VH3 1.7 1.5 1.5 VL1/VH4 1.6 1.8 1.2 VL1/VH5 1.92.3 1.2 VL2/VH2 1.4 2.4 2.3 VL3/VH2 1.9 0.9 2.1 VL3/VH4 1.0 2.3 2.0*Note: This variant corresponds to a non-humanized construct based onthe original clone 390 without the His-tag. Therefore this construct isexactly in the same format as the humanized variants (DKTHT at theC-terminus of HC) mentioned in the same table.

C. Inhibition of Collagen Induced Platelet Aggregation by anti GPVI FabFragments (Human Whole Blood)

For the experiments blood was anticoagulated with 20 μg/ml hirudin andused immediately. Before measurement whole blood was diluted 1:1 withNaCl. Fab fragments were used at final concentration of 0.15-20 μg/mland incubated with blood for 5 min at 37° C. Thereafter collagen wasadded at a concentration of 1 μg/ml and the aggregation response wasmonitored by the measurement of the impedance using Multiplate®analyzer. The reaction was monitored for 6 min. The aggregation responsewas quantified by the area under the aggregation curve (AUC) asspecified by the manufacturer. As shown in FIG. 6 investigated Fabfragments were able to inhibit collagen induced platelet aggregation ina concentration dependent manner.

D. Inhibition of Thrombus Formation Under Flow by anti GPVI FabFragments

For the experiments blood was anticoagulated with 20 μg/ml hirudin andused immediately. Rectangular capillary glass microslides with an innerdiameter of 1×0.1 mm (Camlab, Cambridge, UK) were coated overnight with100 μg/ml Horm collagen and blocked with heat-inactivated 0.5% fattyacid-free BSA at room temperature for 1 h. Blood platelets werefluorescently labeled with 2 μM DiOC6(3) and treated with Fab fragmentsfor 5 min at 37° C. Perfusion through the collagen coated coverslip wasperformed for 2 minutes at 2000 s-1. After blood perfusion, Tyrodesbuffer 1 was perfused through the microslides for 5 min at the sameshear rate. Thrombus formation was quantified by determination ofplatelet (thrombus) surface coverage. For this purpose ten finalfluorescence images were recorded from different areas in the middle ofthe capillary. Additionally, phase contrast and DIC pictures wererecorded. Imaging recording and analysis was performed using ImageProplus imaging software (Mediacy, Silver Spring, USA) connected to ablack-and-white CCD camera (CoolSnap cf, Ropers ScientificGmbH/Photometrics, Ottobrunn). FIG. 7A-E show examples of inhibition ofthrombus formation under flow by anti GPVI-Fab fragments.

E. Effect of the anti-GPVI Fab-Fragment in a Mouse Model of ArterialThrombosis

For this in vivo investigation, mice humanized for the GPVI receptorhave been used. The Carotid artery occlusion was photochemically inducedresulting in vascular endothelial injury at the inner vessel sidewithout affecting the outer vessel wall. By this technique the red dye,rose bengal, is systemically administered and the endothelium of thecarotid artery was irradiated by green laser light resulting in an“inside-out” injury. The anti GPVI Fab-fragment was administered at 10mg/kg as an intravenous bolus via the jugular vein catheter. After a 15min incubation period the laser light source was placed 12 cm away fromthe carotid artery distal to the flow probe and laser irradiation wasstarted. Blood flow was continuously monitored for an observation periodof 90 min. The measured thrombosis parameters were the length of time tocomplete arterial occlusion following vascular injury (time toocclusion, TTO) and the area under the blood flow curve (AUC).

For thrombosis evaluation, two measured parameters were used: a) time toocclusion (TTO) and b) the area under the blood flow curve. The timefrom thrombotic challenge until vessel occlusion (TTO) was 78 min foranti-GPVI Fab fragment versus 33 min for the control group resulting ina 2.4-fold increase (table 17). The area under the flow curve was2.3-fold increased for the group treated with anti-GPVI Fab fragmentcompared to the control group (table 18). Intravenous bolusadministration of 10 mg/kg anti-GPVI Fab fragment resulted in asignificant antithrombotic effect in the photochemical induced arterialthrombosis model in humanized GPVI mice.

TABLE 17 Thrombosis parameter: Time to occlusion (min) Fab TTO (min)Control group 10 mg/kg iv Mean 32.9 77.9 SEM 1.4 5.0 Lower 95% CI ofmean 29.5 65.0 Upper 95% CI of mean 36.4 90.8 Number of animals 6 6

TABLE 18 Thrombosis parameter: Area under the blood flow curve Fab AUC(au) Control group 10 mg/kg iv Mean 1154 2605 SEM 189.2 515.2 Lower 95%CI 667.1 1280 Upper 95% CI 1640 3929 Number of animals 6 6

F. GPVI Depletion from the Platelet Surface Induced by anti-GPVI FabFragments

For the experiments, blood was anticoagulated with 20 μg/ml hirudin andused immediately. Anti GPVI-Fab fragments were used at indicatedconcentrations. Blood or platelet rich plasma (PRP) samples wereincubated with the Fab fragments for 5 min, 15 min, 1 h and 2 h,respectively. Thereafter samples were fixed using paraformaldehyde andGPVI receptor expression was determined. GPVI density on the plateletsurface was measured using a different, fluorescently labelled anti-GPVIantibody and determined in a flow cytometer (BD LSR II). As a control,it was previously shown that this antibody is able to bind GPVIindependently (and in the presence) of the investigated Fab fragment.

As shown in FIG. 8, the anti-GPVI Fab causes a reduction of GPVI surfaceexpression in a concentration dependent manner. Further experiments showthat 5 min of anti GPVI Fab exposure already caused a significant GPVIdepletion at 10 μg/ml and 2 μg/m (FIG. 9). Also lower concentrations ofthe anti-GPVI Fab were able to decrease the GPVI surface density,although with a delayed time course.

These results support the fact that the anti-GP VI Fab induces the GP VIdepletion at the platelet surface.

G. In vitro and ex vivo Effect of an anti GPVI Fab on Collagen-InducedWhole Blood Aggregation in Cynomolgus Monkey (Macaca fascicularis) withConcomitant Hematology Assessment.

1. Animal Details and Dose Regimen:

Animal studies were conducted in an AAALAC (Association for Assessmentand Accreditation of Laboratory Animal Care)-accredited facilityaccording to the local animal welfare regulations and registered by theveterinary authorities. The species/strain used in the study wascynomolgus monkeys (Macaca fascicularis). Only female monkeys were usedfor the study. Two animals per dose group were studied. Doses studiedincluded phosphate buffered saline (PBS) vehicle control, 0.01, 0.1, 1,and 3 mg/kg of anti GPVI Fab all administered at 2 ml/kg IV bolus. Thebodyweight of the included monkeys ranged between 3.52 and 7.34 kg. Oneof the animals (monkey D) used in the 1 mg/kg dose group did not showany platelet aggregation response at pre-dose sample. Hence, nocalculation of intra-individual relative change of aggregation response(inhibition of aggregation in % compared to pre-dose) over time waspossible and no data is provided.

2. Blood Sampling and Processing for Hematology and Plasma Preparation

Whole blood was collected from healthy conscious single-housedcynomolgus monkeys from the antecubital vein after needle puncture atvarious time intervals before and after drug or vehicle administration(pre-dose, 0.5, 1, 2, 4, 6, 8, 24, 48, 72, 149.5 hours) into tubescontaining 3.13% sodium citrate (Eifelfango, Bad Neuenahr-Ahrweiler,Germany) at 1/10 of the total tube volume after blood sampling. From thePBS vehicle treated group the sampling schedule varied slightly and thefollowing time points were sampled: pre-dose, 0.5, 1, 2, 4, 6, 8, 24,48, 126 hours. Whole blood cell counts with a particular focus onplatelet count were determined to monitor the physiological state ofhematology by using an automated hematology analyzer Scil Vet abc (Scilanimal care Company GmbH, Viernheim, Germany). The remaining bloodsample after whole blood aggregation assays was centrifuged for plasmapreparation from each time point. For plasma preparation blood wascentrifuged at 5000 U/min for 15 min and the supernatant collected in aseparate tube and frozen at −20° C. for analysis of plasma levels ofanti GPVI Fab at a later time point.

3. Measurements of Whole Blood Platelet Aggregation

Whole blood platelet aggregation assays were performed using themultiplate® platelet function analyzer (Dynabyte lnformationssystemeGmbH, Munich, Germany). The agonist used was equine type I collagen(Horm collagen, Nycomed, Munich, Germany) at a final concentration of 1μg/ml. The analysis was performed according to the manufacturesinstruction and percent inhibition of whole blood aggregation wascalculated relative to each individual whole blood aggregation responseat pre-dose. Briefly described, the cartridge was preloaded with 297 μlCaCl₂ and 297 μl of whole blood was added. After five minutes ofequilibration 6 μL of the agonist collagen was added in a 100-foldconcentration and the measurement started. The recording took place for7 min and the result was expressed as area under the curve (AUC,arbitrary unit) over time in minutes. Relative change of AUC over timecompared to the pre-dose value was calculated to percent inhibition ofplatelet aggregation and plotted in a graph.

With a separate set of blood samples from other monkeys out of the samecolony in vitro dose-response measurements were performed to determinean in vitro IC₅₀ for the anti GPVI Fab. Therefore blood samples werehandled in the same way as described above for ex vivo measurement.Briefly, different concentrations of anti GPVI Fab (30, 10, 3, 1, 0.3,0.1, 0.03, 0.01 μg/ml) were added to the CaCl₂/whole blood mixture inthe test cell of the multiplate® analyzer and incubated for 5 minutes.After adding the agonist collagen at 1 μg/ml the measurement was startedand the AUC recorded for 7 min. By plotting the respective dose-responsean IC₅₀ was calculated using an in-house statistical software tool(Speed 2.0 LTS).

4. Measurment of GPVI Receptor Surface Expression

Whole blood was collected from healthy conscious single-housedcynomolgus monkeys from the antecubital vein after needle puncture atvarious time intervals before and after drug or vehicle administrationinto tubes containing 3.13% sodium citrate at 1/10 of the total tubevolume after blood sampling and used immediately. Samples were fixedusing 5% paraformaldehyde and GPVI receptor expression was determined.GPVI density on the platelet surface was measured using a different,fluorescently labelled anti-GPVI antibody and determined in a flowcytometer. As a control, it was previously shown that this antibody isable to bind GPVI independently (and in the presence) of theinvestigated Fab fragment.

5. Results on Whole Blood Platelet Aggregation.

To compare the respective dose regimen, percent inhibition of wholeblood platelet aggregation is calculated relative to the pre-dose valueof each individual animal. The PBS vehicle group revealed up to 67% ofinhibition of platelet aggregation at 6 hours after IV bolusadministration (data not shown). Therefore, at least 80% inhibition ofplatelet aggregation over at least two consecutive time points wasconsidered to be physiologically relevant. Both two low doses of antiGPVI Fab tested (0.01 and 0.1 mg/kg anti GPVI fab) did not show anyrelevant inhibition of whole blood aggregation. At 1 mg/kg, 93% plateletinhibition was reached already at 1 hour after administration and stayedabove 80% up to 24 hours (apart from a slight decrease in effect at 2FIG. 10 A). Inhibition of platelet function subsequently decreased overtime. At 3 mg/kg the inhibition of platelet aggregation was stable atgreater values than 80% during the first 24 hours of observation andsustained until 72 hours with values between 69% and 77% (FIG. 10 B). Inboth higher dose groups tested the platelet function fully recovered atthe last time point (149.5 hrs, FIGS. 10 A and B). Based on this data anED₅₀ was estimated at 0.5 hours post IV bolus administration andrevealed 0.5 mg/kg for the tested anti GPVI Fab.

This experiment demonstrates that anti GPVI Fab inhibit ex vivo wholeblood platelet aggregation in a dose-dependent manner when compared tovehicle using collagen (1 μg/ml).

In a separate set of experiments the in vitro activity of the anti GPVIFab was determined. The calculated IC₅₀ revealed 0.81 μg/ml [0.51; 1.28μg/ml] CV=21.6% (FIG. 11).

6. Hematology Assessment

To monitor the physiological state of hematology during the time-courseof the experiment, whole blood cell counts with a particular focus onplatelet count were determined. Mean platelet counts at pre-dose variedbetween 428×10³/μL in PBS vehicle, 369×10³/μL at 0.01 mg/kg anti GPVIFab, 235>10³/μL at 0.1 mg/kg anti GPVI Fab, 357×10³/μL at 1 mg/kg antiGPVI Fab, and 312×10³/μL at 3 mg/kg anti GPVI Fab (FIG. 12 A-E). Duringthe time-course of the experiment the platelet count did not changesubstantially (i.e. values below 100×10³/μL) and the following plateletcount was determined at 126 hours in the PBS vehicle group: 358×10³/μL(FIG. 12 A). In all anti GPVI Fab treated groups the platelet countdetermined at 149.5 hours revealed the following values: 411×10³/μL at0.01 mg/kg anti GPVI Fab, 329×10³/μL at 0.1 mg/kg anti GPVI Fab,321×10³/μL at 1 mg/kg anti GPVI Fab, and 320×10³/μL at 3 mg/kg anti GPVIFab (FIG. 12 B-E). All other determined hematology parameters,hematocrit, red blood cell count, and hemoglobin were not changedsubstantially during the time-course of the experiment (data not shown).

These data demonstate that the GPVI Fab do not affect the physiologicalstate of hematology.

7. GPVI Receptor Expression

As shown in FIG. 13, before iv administration of the anti GPVI Fab allplatelets were positive for GPVI expression (pre-dose). In blood takenafter drug administration no specific signal for GPVI could be observedon the platelet surfaces (24 h). Beginning at 48 h after drugadministration a new population of platelets arises, which are GPVIpositive. 150 h after drug administration all platelet were againpositive for GPVI receptor expression.

This experiment confirms that GPVI Fabs induce GPVI receptor depletionon platelet and that this effect was reversible.

Example 5 Modification of Fab Fragments Properties by Recognition byAuto-Antibodies.

A—Determination of the Activatory Component in Donor Plasma

To investigate the importance of IgG's present in plasma for theactivatory effect of the anti-GPVI Fab, 51 different blood samples weretested. Samples which have been identified as activatory were depletedof IgG's using protein A.

For the experiments, blood was anticoagulated with 20 μg/ml hirudin andused immediately for the preparation of plasma (centrifugation of bloodsamples for 10 min at 1600 g). Thereafter, plasma was depleted of IgG'sfor 2 h at 4° C. using protein A. Protein A was removed bycentrifugation and platelets were added at a final concentration of2×10E8/ml. Anti GPVI-Fab fragments were used at 20 μg/ml. Plasma sampleswere incubated with the Fab fragments or convulxin (or “cvx”, a GP VIspecific agonist) for 15 min followed by a staining with the FITClabelled Pac-1 antibody (specific for activated GPIIbIIIa, which is aplatelet activation marker) for 30 min. Thereafter, samples were fixedusing paraformaldehyde and Pac-1 labelling of platelet was determined ina flow cytometer (BD LSR II).

As seen in FIG. 14, on Control panel, treatment of plasma with protein Ahad no effect on platelet activation by the GPVI specific agonistconvulxin (cvx). However, FIG. 14, panel A shows that the activatoryeffect of Fab was greatly reduced after IgG depletion, suggesting thatpreformed IgG's are an essential component for this response.

B—Determination of the Expression of Platelet Activation MarkersFollowing Modified anti GPVI-Fab Fragment Exposer

To further investigate the role of plama IgG on activation of plateletactivation through Fab, the different Fab fragments were used which wereidentical in their heavy and light chain (HC and LC) sequence but differin their C-terminal modification on the heavy chain.

The four different molecules used are described below and illustrated inFIG. 15.

Fab: control molecule with no C-terminal additional amino acids on HC(FIG. 15 A)

Fab-His-tag: hasHisHisHisHisHis peptide sequence (SEQ ID NO:35) on HCcorresponding to the natural occurring sequence plus C-terminal His-tag(FIG. 15 B)

Fab-Gly4Ser: has a GlyGlyGlyGlySer peptide sequence (SEQ ID NO:36) on HCcorresponding to the natural occurring sequence plus C-terminalGly4Ser—tag (FIG. 15 C)

Fab-(Gly4Ser)2: has a GlyGlyGlyGlySerGlyGlyGlyGlySer peptide sequence(SEQ ID NO:36) on HC corresponding to the natural occurring sequenceplus C-terminal (Gly4Ser)2—tag (FIG. 15 C)

As illustrated in FIG. 14, panel B, the modified Fab do not induce anyactivatory, even without IgG depletion, suggesting that modified Fab arenot recognized by preformed IgG's.

For the experiments, blood was anticoagulated with 20 μg/ml hirudin andused immediately. Anti GPVI-Fab fragments were used at 20 μg/ml. Bloodsamples (1-51) were incubated with the Fab fragments for 15 min followedby a staining with the FITC labelled Pac-1 antibody (specific foractivated GPIIbIIIa, which is platelet activation marker) for 30 min.Thereafter samples were fixed using paraformaldehyde and Pac-1 labellingof platelet was determined in a flow cytometer (BD LSR II).

The activatory potential of the 4 Fab formats was tested on 51 differentblood samples. Fab with no overhang induced a significant increase inPac-1 binding (defined as>5 fold in 23 samples (corresponds to 42%). Insharp contrast, as seen in Table 19 below, Fabs modified at theC-terminus of the HC were much less active in this test with Fab-His-tagand Fab-Gly4Ser only showing activity on 1 sample and Fab-(Gly4Ser)2(longest C-terminal extension) was not active over the threshold. Thisdifferential activatory pattern is also reflected in the cases of minoractivation (2-5 fold over basal). These results indicated that theactivatory potential of Fab fragments in this assay is not determined bythe antigen binding CDR sequences but appears to reside in the HCC-terminus because the activity greatly reduced by C-terminalmodifications of the HC chain. Thus these results suggest that theC-terminal extremity of Fab were recognized by IgG preexisting inpatients blood, which induces platelet activation.

TABLE 19 Fold increase in Pac-1 binding to platelets incubated with Fabfragments with no overhang, Fab-His-tag, Fab-Gly4Ser and Fab-(Gly4Ser)2.51 different samples (donors) were investigated. Grey field representsample with activation over 5 fold basal values.

1. An antibody Fab fragment that specifically binds to human GPVI andinduces GPVI depletion phenotype.
 2. The antibody Fab fragment of claim1, wherein the antibody Fab fragment binds a conformational epitope ofhuman GPVI and contacts human GPVI residues comprising Ser 43, Arg 67and Asp
 81. 3. The antibody Fab fragment of claim 1, wherein the Fabfragment comprises (a) complementarity determining regions (CDRs) of aheavy chain variable region (HCVR) having amino acid sequences definedby SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20; and (b)complementarity determining regions (CDRs) of a light chain variableregion (LCVR) having amino acid sequences defined by SEQ ID NO: 21, SEQID NO: 22, and SEQ ID NO: 23; and wherein at least 2 amino acid residuesof each CDR can be changed to another amino acid residue withoutdirectly disrupting a contact with a GPVI epitope residue.
 4. Theantibody Fab fragment of any one of claim 1, wherein the antibody Fabfragment comprises a heavy chain of SEQ ID NO.6 and a light chain of SEQID NO.8 or sequences having at least 80% identity with these sequences,as far as the antibody Fab fragment binding specificity is maintained.5. The antibody Fab fragment of claim 3 which is a humanized.
 6. Theantibody Fab fragment of claim 5, wherein the humanized Fab fragmentcomprises a combination of a heavy chain variable region (HCVR) aminoacid sequence and a light chain variable region (LCVR) amino acidsequence, selected from the group consisting of (a) LCVR (SEQ ID NO.15)and HCVR (SEQ ID NO.10); (b) LCVR (SEQ ID NO.15) and HCVR (SEQ IDNO.11); (c) LCVR (SEQ ID NO.15) and HCVR (SEQ ID NO.12); (d) LCVR (SEQID NO.15) and HCVR (SEQ ID NO.13); (e) LCVR (SEQ ID NO.15) and HCVR (SEQID NO.14); (f) LCVR (SEQ ID NO.16) and HCVR (SEQ ID NO.11); (g) LCVR(SEQ ID NO.17) and HCVR (SEQ ID NO.11); and (h) LCVR (SEQ ID NO.17) andHCVR (SEQ ID NO.13).
 7. An engineered Fab fragment comprising acombination of a humanized heavy chain (HC) amino acid sequence and ahumanized light chain (LC) amino acid sequence, wherein the humanizedheavy chain further comprises a c-terminal extension comprisingadditional amino acid residues and wherein the c-terminal extensionprevents recognition by anti-Fab antibodies.
 8. The engineered Fabfragment of claim 7, wherein the c-terminal extension is selected fromthe group consisting of SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37. 9.The engineered Fab fragment of claim 8, wherein the Fab fragment is asdefined in any one of claim
 6. 10. A pharmaceutical compositioncomprising an antibody Fab fragment as defined in claims 6 or 9 and apharmaceutically acceptable carrier or excipients.
 11. A polynucleotideencoding a polypeptide selected from the group consisting in SEQ IDNO.6, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ IDNO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, and SEQID NO.17 or a sequence having at least 80% identity with thesesequences, and wherein the polypeptide retains its binding specificity.12. A method for the preparation of an antibody Fab fragment as definedclaims 6 or 9 comprising the steps of: Culture of a cell line expressionan antibody Fab fragment as defined in claims 6 or 9 Purification of Fabfragment expressed in the culture medium Formulation of the Fab fragmentin a convenient form
 13. (canceled)
 14. A method for prevention ofrecognition of an antibody Fab fragments by preexisting antibodiescomprising masking the c-terminal extremity of the antibody Fab fragmentby addition of a molecule to the c-terminus.
 15. A method for preventionof platelet activation when administering an anti-GPVI Fab comprisingmasking the c-terminal extremity of the antibody Fab fragment byaddition of a molecule to the c-terminus.
 16. A method of treating athrombotic or vascular disease comprising administering an antibody Fabfragment as defined in claims 6 or 9 to a patient in need of suchtreatment.